Gas testing unit and method

ABSTRACT

Apparatuses and associated methods are described for the efficient evaluation of C1-containing substrates, and especially for such evaluation conducted locally, or on-site, at a prospective facility for implementation of a biological conversion process for desired end product using a C1 carbon source. The exact composition of a given, industrial C1-containing substrate, as well as the range in composition fluctuations, are generally difficult to reproduce at a remote facility (e.g., a laboratory or a pilot-scale or demonstration-scale process), as required for the accurate prediction/modeling of commercial performance to justify large capital expenditures for commercial scale-up.

CROSS-REFERENCE TO RELATED APPLICATION

This Application is a Continuation of U.S. application Ser. No.14/919,694 filed on Oct. 21, 2015, now U.S. Pat. No. 10,113,194 B2, thecontents of which are incorporated by reference.

FIELD OF THE INVENTION

Aspects of the invention relate to apparatuses that include separatebioreactor stages for assessing the comparative performance between atest CO-containing substrate and a reference CO-containing substrate.Advantageously, such apparatuses may be housed within a containersuitable for transport (e.g., to where an industrial CO-containing wastegas is produced).

DESCRIPTION OF RELATED ART

Environmental concerns over fossil fuel greenhouse gas (GHG) emissionshave led to an increasing emphasis on renewable energy sources. As aresult, ethanol is rapidly becoming a major hydrogen-rich liquidtransport fuel around the world. Continued growth in the global marketfor the fuel ethanol industry is expected for the foreseeable future,based on the increased emphasis on ethanol production in Europe, Japan,and the United States, as well as several developing nations. Forexample, in the United States, ethanol is used to produce E10, a 10%mixture of ethanol in gasoline. In E10 blends, the ethanol componentacts as an oxygenating agent, improving the efficiency of combustion andreducing the production of air pollutants. In Brazil, ethanol satisfiesapproximately 30% of the transport fuel demand, as both an oxygenatingagent blended in gasoline, and as a pure fuel in its own right. Inaddition, the European Union (EU) has mandated targets, for each of itsmember nations, for the consumption of sustainable transport fuels suchas biomass-derived ethanol.

The vast majority of fuel ethanol is produced via traditionalyeast-based fermentation processes that use crop derived carbohydrates,such as sucrose extracted from sugarcane or starch extracted from graincrops, as the main carbon source. However, the cost of thesecarbohydrate feedstocks is influenced by their value in the marketplacefor competing uses, namely as food sources for both humans and animals.In addition, the cultivation of starch or sucrose-producing crops forethanol production is not economically sustainable in all geographies,as this is a function of both local land values and climate. For thesereasons, it is of particular interest to develop technologies thatconvert lower cost and/or more abundant carbon resources into fuelethanol. In this regard, carbon monoxide (CO) is a major, energy-richby-product of the incomplete combustion of organic materials such ascoal, oil, and oil-derived products. CO-rich waste gases result from avariety of industrial processes. For example, the steel industry inAustralia is reported to produce and release into the atmosphere over500,000 metric tons of CO annually.

More recently, microorganism (bacteria) based process alternatives forproducing ethanol from CO on an industrial scale have become a subjectof commercial interest and investment. The ability of microorganismcultures to grow, with CO being the sole carbon source, was firstdiscovered in 1903. This characteristic was later determined to residein an organism's use of the acetyl coenzyme A (acetyl CoA) biochemicalpathway of autotrophic growth (also known as the Woods-Ljungdahl pathwayand the carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS)pathway). A large number of anaerobic organisms includingcarboxydotrophic, photosynthetic, methanogenic, and acetogenic organismshave since been shown to metabolize CO. Anaerobic bacteria, such asthose from the genus Clostridium, are known to produce ethanol from CO,CO₂, and H₂ via the acetyl CoA biochemical pathway. For example, variousstrains of Clostridium ljungdahlii that produce ethanol from gases aredescribed in WO 00/68407; EP 1117309 A1; U.S. Pat. Nos. 5,173,429;5,593,886; 6,368,819; WO 98/00558; and WO 02/08438. The bacteriumClostridium autoethanogenum sp is also known to produce ethanol fromgases (Abrini et al., ARCHIVES OF MICROBIOLOGY 161: 345-351 (1994)).

Because each enzyme of an organism promotes its designated biologicalconversion with essentially perfect selectivity, microbial synthesisroutes can achieve higher yields with lower energy costs compared toconventional catalytic routes. For example, the energy requirements forseparating byproducts, which result from non-selective side reactions,from the desired products may be reduced. In addition, concerns over thepoisoning of catalysts, due to impurities in the reaction medium, arediminished. Despite these apparent advantages, however, the art mustaddress certain challenges presently associated with the microbialsynthesis of ethanol from CO, particularly in terms of ensuring that theproduction rate is competitive with other technologies. When using CO astheir carbon source, the anaerobic bacteria described above produceethanol by fermentation, but they also produce at least one metabolite,for example, CO₂, methane, n-butanol, and/or acetic acid. The formationof any of these metabolites has the potential to significantly impactproductivity and overall economic viability of a given process, asavailable carbon is lost to the metabolite(s) and the productionefficiency of the desired end product is compromised. In addition,unless a metabolite (e.g., acetic acid) itself has value at the time andplace of the microbial fermentation process, it may pose a wastedisposal problem. Various proposals for addressing the formation ofproducts other than the desired end product in the anaerobicfermentation of CO-containing gases to make ethanol are discussed inWO2007/117157, WO2008/115080, and WO2009/022925.

Ethanol production rate, which is a key determinant as to whether agiven fermentation process is economically attractive, is highlydependent on managing the appropriate conditions for bacterial growth.For example, it is known from WO2010/093262 that the CO-containingsubstrate must be provided to a microbial culture at a rate that resultsin optimal microbial growth and/or desired metabolite production. Ifinsufficient substrate is provided, microbial growth slows, and thefermentation product yields shift toward acetic acid at the expense ofethanol. If excessive substrate is provided, poor microbial growthand/or cell death can result. Further information regarding therelationships among operating parameters in these processes is found inWO2011/002318.

The art pertaining to biological processes for producing ethanol fromCO, and particularly CO-containing waste streams such as the gaseouseffluents emitted in steel production and in the chemical industry ingeneral, is continually seeking solutions that improve overall processeconomics (and therefore industry competitiveness), and/or that lead togreater certainty in the adoption of relatively new technologies on anindustrial scale. In this regard, the commercial performance of a givenbacterial culture can be sensitive to the specific source of theCO-containing substrate, and, more particularly, the types and amountsof impurities that may reside in gaseous waste streams of a specificindustrial operator (e.g., steel producer), in addition to variations ingas composition. The large investment for a commercial biologicalconversion process is a difficult financial commitment to undertake ifthe perceived risks associated with an untested, local CO-containingsubstrate and utilities (e.g., water source) are considered excessive.Efficient means of achieving client/investor confidence in a giventechnology are therefore of great importance in advancing biologicalconversion processes for ethanol production to a commercial reality.

SUMMARY OF THE INVENTION

The present invention is associated with the discovery of apparatusesand associated methods for the efficient evaluation of C1-containingsubstrates, and especially for such evaluation that is conductedlocally, or on-site, at a prospective facility for implementation of abiological conversion process for ethanol production from a C1 carbonsource. Typically, the C1-containing substrate comprises at least one C1carbon source selected from the group consisting of CO, CO₂, and CH₄.Importantly, it has been determined that the precise composition of agiven, industrial C1-containing substrate is often difficult toreproduce at a remote facility (e.g., a laboratory or a pilot-scale ordemonstration-scale process), at least to the extent required for theaccurate prediction of commercial performance. Importantly, withoutsufficient confidence that a given process can achieve its performanceobjectives, large capital expenditures needed for scale-up (e.g.,process design and engineering) cannot be justified. In this regard,even trace amounts of certain contaminants (e.g., hydrocarbons orheteroatom-containing hydrocarbons) can adversely affect a bacterialculture, which is a liquid-based system that is prone to extract suchheavier molecules from the C1-containing substrate, allowing suchmolecules to accumulate in internal and external liquid recycle loops ofa bioreactor. Moreover, fluctuations in the local gas composition aresimilarly difficult to reproduce in an off-site testing facility, and inmany cases, the extent of such fluctuations cannot be known orappreciated without direct, local access to the C1-containing substrate.Furthermore, the suitability of other aspects that may be significant tothe locality of a prospective, commercial biological conversion facility(e.g., a local water source to be used in the bacterial culture medium)should be further evaluated and confirmed, prior to significantinvestment decisions.

Advantageously, apparatuses and methods described herein can be used toidentify and remediate causes of sub-optimal performance (e.g.,metabolite productivity and/or substrate utilization). The degree towhich pretreatment of the C1-containing substrate and/or other locallysourced additives to the process must be implemented, or enhanced, canadvantageously be determined in advance of commercial-scale operation,improving the accuracy of the commercial design and associated costestimates. Furthermore, an on-site demonstration of efficacy provides animportant degree of reassurance to both the provider and user alike, ofa prospective biological conversion process, operating with the local(i.e., the actual or industrial) supply of C1-containing substrate andpossibly other local additives.

Particular embodiments of the invention are directed to gas testingunits comprising two bioreactor stages, and in many cases using only twobioreactor stages, with sufficient instrumentation, process equipment,and analytical capability for comparatively evaluating a testC1-containing substrate, and, importantly, with sufficient sizeconstraints to allow transportability.

In one aspect, the present disclosure provides a gas testing unit,comprising: (a) a first bioreactor stage for evaluating the performanceof a reference C1-containing substrate; (b) a second bioreactor stagefor evaluating the performance of a test C1-containing substrate; and(c) an analytical section configured for analysis of both gaseous andliquid products of the first and second bioreactors; wherein the gastesting unit is capable of being housed within a container having avolume of less than about 6 m³ and transportable to multiple locations.

The gas testing unit is capable of being housed within a box havinglength, width, and height dimensions of less than about 1.8 meters each,or less than about 1.6 meters each, or less than 1.3 meters each. Incertain embodiments, the box has one of the length, width, and heightdimensions of less than about 1.6 meters, and the other two of thelength, width, and height dimensions of less than about 1.3 meters.

The analytical section of the gas testing system comprises a gaschromatography (GC) analyzer having first and second chromatographycolumns configured, respectively, for analysis of the gaseous and theliquid products.

The bioreactors of the first and second bioreactor stages each comprisea circulated loop bioreactor. The first and second bioreactor stageseach further comprise external recycle loops and recirculation pumps forrecycling liquid withdrawn proximate bottom ends of the bioreactors toproximate, opposite top ends of the bioreactors.

The gas testing unit may further comprise an operating control systemfor controlling one or more operating parameters selected from the groupconsisting of fresh culture medium addition rate, gaseous C1-containingsubstrate feed rate, reactor temperature, and reactor pH. In certainembodiments, the one or more operating parameters include reactor pH,and the control system includes instrumentation for controlling the flowof a basic neutralizing agent to the bioreactor, based on a measuredreactor pH.

In certain embodiments, the gas testing unit comprises a safety controlsystem for suspending the flow of at least the test C1-containingsubstrate or the reference C1-containing substrate, in response to ameasurement of an ambient C1 concentration at above a thresholdconcentration.

In a second aspect, the present disclosure provides a method forevaluating suitability of a test C1-containing substrate for use in abiological conversion process, the method comprising (a) feeding areference C1-containing substrate to a first bioreactor containing afirst culture of a C1-fixing microorganism; (b) feeding the testC1-containing substrate to a second bioreactor containing a secondculture of the C1-fixing microorganism; and (c) analyzing both gaseousand liquid products of the first and second bioreactors to determine theperformance of the first and second bioreactors; wherein the suitabilityof the test C1-containing substrate is established from a comparison ofthe performance of the first bioreactor, relative to the performance ofthe second bioreactor. In certain embodiments, at least a portion ofstep (a) and step (b) are carried out simultaneously.

In certain embodiments, the C1-fixing microorganism is acarboxydotrophic microorganism from the genus Clostridium. Preferably,the C1-fixing microorganism is selected from the group consisting ofClostridium autoethanogenum, Clostridium ljungdahlii, and Clostridiumragsdalei.

In certain embodiment, the method comprises feeding the referenceC1-containing substrate to the second bioreactor, prior to feeding thetest C1-containing substrate to the second bioreactor in step (b).

The test C1-containing substrate is an industrial C1-containing wastegas stream that has been pretreated to remove a contaminant. In certainembodiments, the test C1-containing substrate is a raw industrial gasstream. In certain embodiments, the method comprises testing a rawC1-containing substrate to determine the biological process is feasibleon an untreated waste gas stream.

In one embodiment, the analyzing step (c) comprises measuringconcentrations of C1 in the gaseous products of the first and secondbioreactors and measuring concentrations of ethanol and at least onefurther metabolite in the liquid products of the first and secondbioreactors. Additionally, in accordance with the invention at least oneof the first and second cultures of a C1-fixing microorganism maycomprise a culture medium prepared with, or supplemented with, a localwater source. In some embodiments, the performances of the first andsecond cultures are assessed simultaneously over a test period of atleast about 7 days

In a further aspect, the present disclosure provides a method fordetermining whether a test C1-containing substrate supports a biologicalconversion process. The method comprises: (a) maintaining separate,first and second cultures of a C1-fixing microorganism, utilizing areference C1-containing substrate as a nutrient for producing ethanoland at least one further metabolite; (b) changing from the referenceC1-containing substrate, as the nutrient to the second culture, to atest C1-containing substrate; (c) assessing the performance of the firstculture, relative to that of the second culture, under a same set oftarget operating conditions, but using the different, reference and testC1-containing substrates; (d) in the event of not obtaining a minimumperformance deficit of the second culture in step (c), confirming thatthe test C1-containing substrate supports the biological conversionprocess; (e) in the event of obtaining the minimum performance deficitin step (c), pretreating or enhancing pretreatment of the testC1-containing substrate to provide a higher quality test C1-containingsubstrate, relative to the test C1-containing substrate used to assessperformance in step (c).

In one embodiment, the method further comprises (f) assessing theperformance of the first culture, relative to that of a third culture,under the same set of target operating conditions, but using thedifferent, reference and higher quality test C1-containing substrates;and (g) in the event of not obtaining the minimum performance deficit ofthe third culture in step (f), confirming that the higher quality testC1-containing substrate supports a biological conversion process.

In certain embodiments, different water sources are used to prepare thefirst and second cultures or supplement the first and second cultures.

These and other embodiments, aspects, and advantages relating to thepresent invention are apparent from the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the exemplary embodiments of thepresent invention and the advantages thereof may be acquired byreferring to the following description in consideration of theaccompanying figures, in which similar features are identified bysimilar reference numbers (e.g., bioreactor 100 of FIG. 1A andbioreactors 100 a, 100 b of FIG. 2).

FIGS. 1A and 1B depict sectional side and rear views, respectively, ofrepresentative, transportable gas testing units as described herein.

FIG. 2 depicts a close-up view of representative bioreactors for use ingas testing units as described herein and provides additional detailsrelating to their operation.

FIG. 3 is a flowchart illustrating a representative methodology, whichmay be performed with gas testing units as described herein, fordetermining whether a test C1-containing substrate, optionally followingone or more remedial measures as described herein (e.g., increasing itspurity), is suitable for a biological conversion process.

FIGS. 1-3 should be understood to present an illustration of thedisclosure and/or principles involved. In order to facilitateexplanation and understanding, simplified equipment and process flowsare depicted in FIGS. 1 and 2, and the relative dimensions of differentequipment are not necessarily drawn to scale. Details including somevalves, instrumentation, and other equipment and systems not essentialto the understanding of the disclosure are not shown. As is readilyapparent to one of skill in the art having knowledge of the presentdisclosure, apparatuses, and methods for testing whether a givenC1-containing substrate and/or local additives support a biologicalconversion process will have configurations and components determined,in part, by their specific use.

DETAILED DESCRIPTION

The invention is associated with the important recognition that a test(or local) C1-containing substrate can be effectively evaluated, for thepurposes identified above, using only a selected portion of theequipment otherwise used for implementing a biological C1 conversionprocess with maximum productivity and yield of a desired product. The C1containing substrate typically comprises at least one C1 carbon sourceselected from the group consisting of CO, CO₂, and CH₄. For example, theC1 containing substrate may be a gaseous substrate containing CO. The C1containing substrate may also comprise H₂ and/or N₂. For example, aparallel bioreactor stage system, with separate, first and secondbioreactors for comparative testing of a test gas and a reference gas,can provide the necessary information to confirm that the locallyavailable gas feed and/or additives support a commercial process, evenwithout reaching commercial levels of performance (e.g., in terms ofliquid product ethanol titers). Only a subset of the actual bioreactorcomponents, process vessels, instrumentation, and analyzers arerequired, making it possible for representative gas testing units to behoused and transported (e.g., in the cargo bay of a 747 jetliner) to theprospective facility. Particular efficiencies may be gained, forexample, by having only two bioreactors for evaluating test andreference gases, respectively, with no reactor internal distributiondevices, except optionally liquid distribution devices (e.g., showerheads) for feeding liquid to the tops of the bioreactors from externalrecycle loops. Other efficiencies may be gained from using gaschromatography (GC) for analysis of both gaseous and liquid products.Yet further efficiencies may be gained by avoiding, at each bioreactorstage, the separation and recycling of a C1-fixing microorganism. Byexploiting these and other efficiencies, gas testing units mayadvantageously be made transportable (e.g., by air, sea, or land) to aremote site of a prospective, commercial-scale installation of abiological conversion process for producing ethanol from a C1-containingsubstrate. The gas testing units include sufficient equipment foron-site evaluation of the locally available C1-containing substrate andprocess additives such as water but without all of the requirements of(i) reactor systems needed for productivity maximization, and/or (ii)analytical systems and instrumentation for comprehensive monitoring andcontrol of all process variables. Such requirements are generally notaligned with the objective of transportability. Advantageously, it hasbeen determined that qualitative results (e.g., in comparison with areference test), as opposed to quantitative results, can provide ameaningful evaluation for gas quality validation purposes and/oridentify areas in which a remedial measure is necessary to address aperformance deficit.

The present invention relates to gas testing units that operate by, andassociated methods that otherwise involve, the production of a desiredend product, such as ethanol, from the biological conversion of a C1carbon source in a gaseous C1-containing substrate. First and secondbioreactor stages of the gas testing units can be fed, for example, witha reference (or control) C1-containing substrate and a test (orindustrially available) C1-containing substrate for parallel orsimultaneous performance evaluation, in order to establish a comparisonthat provides useful information in terms of establishing that aspecific, test C1-containing substrate is suitable for a given process.Each of the bioreactor stages comprises a bioreactor that, in operation,includes a liquid culture medium containing a C1 fixing microorganism(bacterial culture). In addition to the desired end product, thebiological conversion processes, occurring in each of the bioreactorstages, additionally generate undesired or less desired metabolites,which, like the desired product (e.g., ethanol), can be detected inliquid products withdrawn from these stages. Examples of suchmetabolites are acetate (e.g., in the form of acetic acid) and2,3-butanediol. The terms “acetate” or “acetic acid” refer to the totalacetate present in the culture medium, either in its anionic(dissociated) form (i.e., as acetate ion or CH₃COO⁻) or in the form offree, molecular acetic acid (CH₃COOH), with the ratio these forms beingdependent upon the pH of the system. As described below, a basicneutralizing agent such as aqueous ammonium hydroxide (NH₄OH) or aqueoussodium hydroxide (NaOH) may be used to control the pH of the culturemedium in a given bioreactor (e.g., to a pH setpoint value that may beany specific pH value between pH=4.5 and pH=8.0), by neutralizing theformed acetic acid. Representative pH ranges at which bioreactors aremaintained (or controlled) for carrying out the processes describedherein are generally any pH value (set point) within the range fromabout 4.0 to about 8.0, such as from about 5.0 to about 6.5 (e.g.,pH=5.0, 5.5, or 6.0).

Representative C1-fixing bacterium, are those from the genus Moorella,Clostridia, Ruminococcus, Acetobacterium, Eubacterium, Butyribacterium,Oxobacter, Methanosarcina, Methanosarcina, and Desulfotomaculum.

A “microorganism” is a microscopic organism, especially a bacterium,archea, virus, or fungus. The microorganism of the invention istypically a bacterium. As used herein, recitation of “microorganism”should be taken to encompass “bacterium.”

The microorganism of the invention may be further classified based onfunctional characteristics. For example, the microorganism of theinvention may be or may be derived from a C1-fixing bacterium, ananaerobe, an acetogen, an ethanologen, a carboxydotroph, and/or amethanotroph. Table 1 provides a representative list of microorganismsand identifies their functional characteristics.

TABLE 1 C1-fixing Anaerobe Acetogen Ethanologen Autotroph CarboxydotrophMethanotroph Acetobacterium woodii + + + +/−¹ − +/−² − Alkalibaculumbacchii + + + + + + − Blautia product + + + − + + − Butyribacteriummethylotrophicum + + + + + + − Clostridium aceticum + + + − + + −Clostridium autoethanogenum + + + + + + − Clostridiumcarboxydivorans + + + + + + − Clostridium coskatii + + + + + + −Clostridium drakei + + + − + + − Clostridium formicoaceticum + + + − + +− Clostridium ljungdahlii + + + + + + − Clostridium magnum + + + − ++/−³ − Clostridium ragsdalei + + + + + + − Clostridiumscatologenes + + + − + + − Eubacterium limosum + + + − + + − Moorellathermautotrophica + + + + + + − Moorella thermoacetica (formerly + + +−⁴ + + − Clostridium thermoaceticum) Oxobacter pfennigii + + + − + + −Sporomusa ovata + + + − + +/−⁵ − Sporomusa silvacetica + + + − + +/−⁶ −Sporomusa sphaeroides + + + − + +/−⁷ − Thermoanaerobacter kiuvi + + +− + − − ¹ Acetobacterium woodii can produce ethanol from fructose, butnot from gas. ²It has been reported that Acetobacterium woodii can growon CO, but the methodology is questionable. ³It has not beeninvestigated whether Clostridium magnum can grow on CO. ⁴One strain ofMoorella thermoacetica, Moorella sp. HUC22-1 has been reported toproduce ethanol from gas. ⁵It has not been investigated whetherSporomusa ovata can grow on CO. ⁶It has not been investigated whetherSporomusa silvacetica can grow on CO. ⁷It has not been investigatedwhether Sporomusa sphaeroides can grow on CO.

“C1” refers to a one-carbon molecule, for example, CO, CO₂, CH₄, orCH₃OH. “C1-oxygenate” refers to a one-carbon molecule that alsocomprises at least one oxygen atom, for example, CO, CO₂, or CH₃OH.“C1-carbon source” refers a one carbon-molecule that serves as a partialor sole carbon source for the microorganism of the invention. Forexample, a C1-carbon source may comprise one or more of CO, CO₂, CH₄.Preferably, the C1-carbon source comprises one or both of CO and CO₂. A“C1-fixing microorganism” is a microorganism that has the ability toproduce one or more products from a C1-carbon source. Typically, themicroorganism of the invention is a C1-fixing bacterium. In a preferredembodiment, the microorganism of the invention is derived from aC1-fixing microorganism identified in Table 1.

An “anaerobe” is a microorganism that does not require oxygen forgrowth. An anaerobe may react negatively or even die if oxygen ispresent above a certain threshold. Typically, the microorganism of theinvention is an anaerobe. In a preferred embodiment, the microorganismof the invention is derived from an anaerobe identified in Table 1.

An “acetogen” is a microorganism that produces or is capable ofproducing acetate (or acetic acid) as a product of anaerobicrespiration. Typically, acetogens are obligately anaerobic bacteria thatuse the Wood-Ljungdahl pathway as their main mechanism for energyconservation and for the synthesis of acetyl-CoA and acetyl-CoA-derivedproducts, such as acetate (Ragsdale, Biochim Biophys Acta, 1784:1873-1898, 2008). Acetogens use the acetyl-CoA pathway as a (1)mechanism for the reductive synthesis of acetyl-CoA from CO₂, (2)terminal electron-accepting, energy conserving process, (3) mechanismfor the fixation (assimilation) of CO₂ in the synthesis of cell carbon(Drake, Acetogenic Prokaryotes, In: The Prokaryotes, 3rd edition, p.354, New York, N.Y., 2006). All naturally occurring acetogens areC1-fixing, anaerobic, autotrophic, and non-methanotrophic. Typically,the microorganism of the invention is an acetogen. In a preferredembodiment, the microorganism of the invention is derived from anacetogen identified in Table 1.

An “ethanologen” is a microorganism that produces or is capable ofproducing ethanol. Typically, the microorganism of the invention is anethanologen. In a preferred embodiment, the microorganism of theinvention is derived from an ethanologen identified in Table 1.

An “autotroph” is a microorganism capable of growing in the absence oforganic carbon. Instead, autotrophs use inorganic carbon sources, suchas CO and/or CO₂. Typically, the microorganism of the invention is anautotroph. In a preferred embodiment, the microorganism of the inventionis derived from an autotroph identified in Table 1.

A “carboxydotroph” is a microorganism capable of utilizing CO as a solesource of carbon. Typically, the microorganism of the invention is acarboxydotroph. In a preferred embodiment, the microorganism of theinvention is derived from a carboxydotroph identified in Table 1.

A “methanotroph” is a microorganism capable of utilizing methane as asole source of carbon and energy. In certain embodiments, themicroorganism of the invention is derived from a methanotroph.

More broadly, the microorganism of the invention may be derived from anygenus or species identified in Table 1.

In a preferred embodiment, the microorganism of the invention is derivedfrom the cluster of Clostridia comprising the species Clostridiumautoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei.These species were first reported and characterized by Abrini, ArchMicrobiol, 161: 345-351, 1994 (Clostridium autoethanogenum), Tanner, IntJ System Bacteriol, 43: 232-236, 1993 (Clostridium ljungdahlii), andHuhnke, WO 2008/028055 (Clostridium ragsdalei).

These three species have many similarities. In particular, these speciesare all C1-fixing, anaerobic, acetogenic, ethanologenic, andcarboxydotrophic members of the genus Clostridium. These species havesimilar genotypes and phenotypes and modes of energy conservation andfermentative metabolism. Moreover, these species are clustered inclostridial rRNA homology group I with 16S rRNA DNA that is more than99% identical, have a DNA G+C content of about 22-30 mol %, aregram-positive, have similar morphology and size (logarithmic growingcells between 0.5-0.7×3-5 μm), are mesophilic (grow optimally at 30-37°C.), have similar pH ranges of about 4-7.5 (with an optimal pH of about5.5-6), lack cytochromes, and conserve energy via an Rnf complex. Also,reduction of carboxylic acids into their corresponding alcohols has beenshown in these species (Perez, Biotechnol Bioeng, 110:1066-1077, 2012).Importantly, these species also all show strong autotrophic growth onCO-containing gases, produce ethanol and acetate (or acetic acid) asmain fermentation products, and produce small amounts of 2,3-butanedioland lactic acid under certain conditions.

However, these three species also have a number of differences. Thesespecies were isolated from different sources: Clostridiumautoethanogenum from rabbit gut, Clostridium ljungdahlii from chickenyard waste, and Clostridium ragsdalei from freshwater sediment. Thesespecies differ in utilization of various sugars (e.g., rhamnose,arabinose), acids (e.g., gluconate, citrate), amino acids (e.g.,arginine, histidine), and other substrates (e.g., betaine, butanol).Moreover, these species differ in auxotrophy to certain vitamins (e.g.,thiamine, biotin). These species have differences in nucleic and aminoacid sequences of Wood-Ljungdahl pathway genes and proteins, althoughthe general organization and number of these genes and proteins havebeen found to be the same in all species (Kopke, Curr Opin Biotechnol,22: 320-325, 2011).

Thus, in summary, many of the characteristics of Clostridiumautoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei arenot specific to that species but are rather general characteristics forthis cluster of C1-fixing, anaerobic, acetogenic, ethanologenic, andcarboxydotrophic members of the genus Clostridium. However, since thesespecies are, in fact, distinct, the genetic modification or manipulationof one of these species may not have an identical effect in another ofthese species. For instance, differences in growth, performance, orproduct production may be observed.

The microorganism of the invention may also be derived from an isolateor mutant of Clostridium autoethanogenum, Clostridium ljungdahlii, orClostridium ragsdalei. Isolates and mutants of Clostridiumautoethanogenum include JA1-1 (DSM10061) (Abrini, Arch Microbiol, 161:345-351, 1994), LBS1560 (DSM19630) (WO 2009/064200), and LZ1561(DSM23693). Isolates and mutants of Clostridium ljungdahlii include ATCC49587 (Tanner, Int J Syst Bacteriol, 43: 232-236, 1993), PETCT(DSM13528, ATCC 55383), ERI-2 (ATCC 55380) (U.S. Pat. No. 5,593,886),C-01 (ATCC 55988) (U.S. Pat. No. 6,368,819), 0-52 (ATCC 55989) (U.S.Pat. No. 6,368,819), and OTA-1 (Tirado-Acevedo, Production of bioethanolfrom synthesis gas using Clostridium ljungdahlii, PhD thesis, NorthCarolina State University, 2010). Isolates and mutants of Clostridiumragsdalei include PI 1 (ATCC BAA-622, ATCC PTA-7826) (WO 2008/028055).

The microorganism of the invention may be cultured to produce one ormore products. For instance, Clostridium autoethanogenum produces or canbe engineered to produce ethanol (WO 2007/117157), acetate (WO2007/117157), butanol (WO 2008/115080 and WO 2012/053905), butyrate (WO2008/115080), 2,3-butanediol (WO 2009/151342), lactate (WO 2011/112103),butene (WO 2012/024522), butadiene (WO 2012/024522), methyl ethyl ketone(2-butanone) (WO 2012/024522 and WO 2013/185123), ethylene (WO2012/026833), acetone (WO 2012/115527), isopropanol (WO 2012/115527),lipids (WO 2013/036147), 3-hydroxypropionate (3-HP) (WO 2013/180581),isoprene (WO 2013/180584), fatty acids (WO 2013/191567), 2-butanol (WO2013/185123), 1,2-propanediol (WO 2014/0369152), and 1-propanol (WO2014/0369152). In addition to one or more target products, themicroorganism of the invention may also produce ethanol, acetate, and/or2,3-butanediol. In certain embodiments, microbial biomass itself may beconsidered a product.

Generally, the same microorganisms are used in the first and secondbioreactors; however, it is also possible in some embodiments to usedifferent C1-fixing microorganisms in the different bioreactors.

Representative C1 containing substrates and particularly the test C1containing substrates as described herein include broadly any C1-carbonsource. A C1-carbon source refers a one carbon-molecule that serves as apartial or sole carbon source for the microorganism of the invention.For example, a C1-carbon source may comprise one or more of CO, CO₂, orCH₄. Preferably, the C1-carbon source comprises one or both of CO andCO₂. The substrate may further comprise other non-carbon components,such as H₂, N₂, or electrons.

The C1 containing substrate may contain a significant proportion of CO,preferably at least about 5% to about 99.5% CO by volume. Suchsubstrates are often produced as waste products of industrial processessuch as steel manufacturing processes or non-ferrous productmanufacturing process. Other processes in which gaseous CO-containingsubstrates are generated include petroleum refining processes, biofuelproduction processes (e.g., pyrolysis processes and fattyacid/triglyceride hydroconversion processes), coal and biomassgasification processes, electric power production processes, carbonblack production processes, ammonia production processes, methanolproduction processes, and coke manufacturing processes. A number ofchemical industry effluents, as well as syngases (containing both CO andH₂) produced from a variety of substrates, can likewise serve aspotential CO-containing substrates. Specific examples include effluentsfrom the production of phosphate and chromate. Advantageously, wastes(e.g., waste gases) from these processes may be used as described hereinfor the beneficial production of useful end products such as ethanol.The substrate and/or C1-carbon source may be or may be derived from awaste or off-gas obtained as a byproduct of an industrial process orfrom some other source, such as from automobile exhaust fumes or biomassgasification. In certain embodiments, the industrial process is selectedfrom the group consisting of ferrous metal products manufacturing, suchas a steel mill manufacturing, non-ferrous products manufacturing,petroleum refining processes, coal gasification, electric powerproduction, carbon black production, ammonia production, methanolproduction, and coke manufacturing. In these embodiments, the substrateand/or C1-carbon source may be captured from the industrial processbefore it is emitted into the atmosphere, using any convenient method.

The substrate and/or C1-carbon source may be or may be derived fromsyngas, such as syngas obtained by gasification of coal or refineryresidues, gasification of biomass or lignocellulosic material, orreforming of natural gas. In another embodiment, the syngas may beobtained from the gasification of municipal solid waste or industrialsolid waste.

In connection with substrates and/or C1-carbon sources, the term“derived from” refers to a substrate and/or C1-carbon source that issomehow modified or blended. For example, the substrate and/or C1-carbonsource may be treated to add or remove certain components or may beblended with streams of other substrates and/or C1-carbon sources.

The composition of the substrate may have a significant impact on theefficiency and/or cost of the reaction. For example, the presence ofoxygen (O₂) may reduce the efficiency of an anaerobic fermentationprocess. Depending on the composition of the substrate, it may bedesirable to treat, scrub, or filter the substrate to remove anyundesired impurities, such as toxins, undesired components, or dustparticles, and/or increase the concentration of desirable components.

The substrate generally comprises at least some amount of CO, such asabout 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mol % CO. Thesubstrate may comprise a range of CO, such as about 20-80, 30-70, or40-60 mol % CO. Preferably, the substrate comprises about 40-70 mol % CO(e.g., steel mill or blast furnace gas), about 20-30 mol % CO (e.g.,basic oxygen furnace gas), or about 15-45 mol % CO (e.g., syngas). Insome embodiments, the substrate may comprise a relatively low amount ofCO, such as about 1-10 or 1-20 mol % CO. The microorganism of theinvention typically converts at least a portion of the CO in thesubstrate to a product. In some embodiments, the substrate comprises noor substantially no CO.

The substrate may comprise some amount of H₂. For example, the substratemay comprise about 1, 2, 5, 10, 15, 20, or 30 mol % H₂. In someembodiments, the substrate may comprise a relatively high amount of H₂,such as about 60, 70, 80, or 90 mol % Hz. In further embodiments, thesubstrate comprises no or substantially no H₂.

The substrate may comprise some amount of CO₂. For example, thesubstrate may comprise about 1-80 or 1-30 mol % CO₂. In someembodiments, the substrate may comprise less than about 20, 15, 10, or 5mol % CO₂. In another embodiment, the substrate comprises no orsubstantially no CO₂.

Although the substrate is typically gaseous, the substrate may also beprovided in alternative forms. For example, the substrate may bedissolved in a liquid saturated with a CO-containing gas using amicrobubble dispersion generator. By way of further example, thesubstrate may be adsorbed onto a solid support.

It has been determined that the precise composition of a given,industrial C1-containing substrate is often difficult to reproduce at aremote facility (e.g., a laboratory or a pilot-scale ordemonstration-scale process), at least to the extent required for theaccurate prediction of commercial performance. Importantly, withoutsufficient confidence that a given process can achieve its performanceobjectives, large capital expenditures needed for scale-up (e.g.,process design and engineering) cannot be justified. In this regard,even trace amounts of certain contaminants (e.g., hydrocarbons orheteroatom-containing hydrocarbons) can adversely affect a bacterialculture, which is a liquid-based system that is prone to extract suchheavier molecules from the C1-containing substrate, allowing suchmolecules to accumulate in internal and external liquid recycle loops ofa bioreactor. Moreover, fluctuations in the local gas composition aresimilarly difficult to reproduce in an off-site testing facility, and inmany cases, the extent of such fluctuations cannot be known orappreciated without direct, local access to the C1-containing substrate.Furthermore, the suitability of other aspects that may be significant tothe locality of a prospective, commercial biological conversion facility(e.g., a local water source to be used in the bacterial culture medium)should be further evaluated and confirmed, prior to significantinvestment decisions.

The use of industrial C1 containing substrates in a biologicalconversion process has been shown to present numerous challenges. Thepresence of substances other than the primary gas components (such asCO, H₂, N₂, CO₂) that may have a detrimental impact on the fermentationprocess. Furthermore, the flow rate of the gas from an industrialprocess is dependent on the operating parameters of that process and isnot tailored to provide a consistent volumetric gas feed rate (e.g. inNm³/hr.) to a downstream fermentation process. The chemistry of the gasin terms of the relative amounts of each of the constituents (bothprimary components and contaminants) change, often rapidly, with timeaccording to the operating parameters and inputs to the upstreamindustrial process.

Perhaps the most significant challenge to the use of industrial off gasas the sole carbon and energy feedstock to a gas fermentation processfor product synthesis is the presence of a broad spectrum ofbactericidal or toxic contaminants. The negative impact of contaminantsfrom industrially produced syngas product of gasified biomass onmicrobial fermentation has been well documented. These gases containboth tars and nitrogen compounds that have been consistentlydemonstrated to inhibit microbial growth and productivity, particularlyamong carboxydotrophic organisms using CO and H₂ as their sole source ofcarbon and energy (Ahmed et al. 2006). Nitric oxide found in syngas hasbeen shown in several studied to be inhibitory to carboxydotrophicorganisms such as C. carboxydivorans and C. ragsdalei at concentrationsas low as 40 ppm (Datar et. al. 2004; Lewis et. al. 2006; Ahmed andLewis, 2007; Kundiyana et. al. 2010. Other studies demonstrated thattars composed of benzene, toluene ethylbenzene and p-xylene (allcompounds found in off-gases from steel making described in table 2)were also found to be inhibitory to the productivity and viability ofcarboxydotrophic organisms (Ahmed et. al 2006; Lewis et. al. 2006).

For example, off-gases produced as an inevitable consequence of thesteel making process contain CO and, in some cases, H₂, and epitomizethe challenge associated with using industrial off gases as describedabove. Typically, waste gases from steel manufacturing processes containlittle or no hydrogen. Further, the multiple contaminating compoundsfound in these off-gases are well known and have been documented (seetable 2). The number and variety of contaminants found in a steeloff-gas stream are certainly much greater than that reported to bepresent in other industrial gases such as biomass-derived syngas. Thisincrease in both the number and variety of contaminants presents a moresignificant challenge to a fermentation system. Although the “additive”effect of contaminants on the biological processes difficult to predictexactly, it is anticipated that the detrimental effect will be much moresevere. Amongst the 15 most abundant contaminants passing into the ventor stacks as part of the off-gas from the steelmaking process arecompounds such as oxides of nitrogen, sulfur dioxide, benzene, toluene,cyanide and fluoride compounds each of which are understood to be toxicto bacteria.

As mentioned above, tars composed of benzene, toluene, ethylbenzene, andp-xylene have been found to have a highly detrimental effect on theviability and productivity of C. carboxydivorans (Lewis et. al. 2006).The relative toxicity of benzene, toluene, and xylene to anaerobicbacteria was described by Payne and Smith (1983). However, as notedabove, this and other compounds are present in steel mill gas togetherwith a variety of other potentially toxic compounds. The additive impactof heavy metals such as cadmium, nickel, and zinc on the toxicity oftoluene was described by (Amor et. al. 2001). These data clearlydemonstrate that microbial performance in the presence of toluene issignificantly and detrimentally impacted by the addition of these heavymetals individually. In steel mill off-gas, these metals are presenttogether, which one would expect would provide an even greater challengeto microbial performance and productivity.

Significantly, it is difficult to provide a test stream in a laboratorysetting that is adequately representative of an industrial gas stream.Importantly, even in gas streams from similar industries, the types andnumber of contaminants present in the individual gas stream will varysignificantly. Even within a single plant or facility, the compositionof the exhaust gas may vary depending on upstream conditions and thesourced raw material provided to the industrial process. Furthermore,compressing gases typically changes the gas composition. In particular,at high pressure, contaminant tend to drop out of the gaseous phase.This causes a discrepancy/variation between the exit gas at the site,and the test sample provided to a laboratory.

Table 2 shows all air emissions (Point source+Fugitive1) in Kilogramsfrom the BlueScope Steel Port Kembla Steelworks—Port Kembla, NSW,Australia as reported in the National Pollution Inventory (NPI)(http://www.npi.gov.au). This emissions data shows the typical pollutioncausing components of off-gases from the BlueScope Steel Port KemblaSteelworks—Port Kembla, NSW, Australia.

TABLE 2 Substance Air Total (kg) Oxides of Nitrogen 7927779 Sulfurdioxide 7498915 Particulate Matter 10.0 um 1722175 Ammonia (total)735551 Sulfuric acid 259163 Total Volatile Organic Compounds 240305Hydrochloric acid 190953 Benzene 130905 Particulate Matter 2.5 um 110063Hydrogen sulfide 81748 Toluene (methylbenzene) 20220 Cyanide (inorganic)compounds 19483 Fluoride compounds 16780 Methanol 12131 Methyl isobutylketone 10775 Zinc and compounds 8228 Manganese & compounds 4001 Chlorine& compounds 3221 Xylenes (individual or mixed isomers) 2583 Lead &compounds 2391 n-Hexane 1142 Styrene (ethenylbenzene) 900 Copper &compounds 575 Cadmium & compounds 425 Nickel & compounds 323 Boron &compounds 247 Polycyclic aromatic hydrocarbons (B[a]Peq) 192 Chromium(III) compounds 176 Mercury & compounds 168 Ethanol 123 1,3-Butadiene(vinyl ethylene) 120 Phenol 115 Selenium & compounds 112 Chromium (VI)compounds 69 Biphenyl (1,1-biphenyl) 60 Arsenic & compounds 47Formaldehyde (methyl aldehyde) 47 Acetone 26 Antimony & compounds 18Ethylbenzene 18 Carbon disulfide 13 Cobalt & compounds 8 Beryllium &compounds 2 Nitric acid 1 Polychlorinated dioxins and furans (TEQ)1.35E−04 ¹Point source emissions flow into a vent or stack and areemitted through a single point source into the atmosphere. Examples arethe exhaust system of a boiler or stationary combustion engine poweredequipment. Fugitive emissions are emissions that are not released via avent or stack. Examples of fugitive emissions include exhaust emissionsfrom vehicles, evaporative emissions from vehicle fuel tanks,volatilization of vapor from vats or fuel and other volatile organicliquid storage tanks, open vessels, spills, and materials handling.Emissions from ridgeline roof vents, louvers and open doors of abuilding, equipment leaks, valve leaks, and flanges are other types offugitive emissions.

As described below, a specific type of bioreactor that is particularlyuseful in the gas testing units and methods described herein is acirculated loop reactor in which the gaseous C1-containing substrate istypically distributed (e.g., sparged) into the bottom of a risersection, at or near the lower end of the reactor containing thebacterial culture medium in a continuous liquid phase. Rising gasbubbles are confined to the riser section during their upward movementthrough the continuous liquid phase until any unconsumed and undissolvedgas is released into a continuous gas phase (i.e., vapor space orheadspace) above the liquid level and extending to the upper end of thereactor. Circulation of the continuous liquid phase in the riser sectionmay be induced by the relatively low density, central portion, throughwhich the majority of the rising gas bubbles pass, in combination withthe relatively high density, peripheral (outer) portion, having littleor no gas holdup. Internal liquid circulation can, therefore, beestablished through net upward movement of the liquid in the centralportion and net downward movement in the peripheral portion. Asdescribed in greater detail below, a bioreactor stage, comprising acirculated loop reactor, may also include forced liquid circulationexternal to the reactor, preferably through the withdrawal of liquidfrom the bottom end of the reactor and introduction of the withdrawnliquid into the top end of the reactor, thereby providing countercurrentgas-liquid flow in the reactor headspace.

The term “bioreactor,” as well as any bioreactor that may be included aspart of a “bioreactor stage,” of a gas testing unit is not limited to acirculated loop reactor, but more broadly includes any suitable vessel,or section within a vessel, for maintaining a liquid volume of culturemedium with a C1-fixing microorganism that may be used to carry out thebiological processes described herein, which may also be referred to asfermentation processes to the extent that they are generally conductedanaerobically. Particular types of bioreactors can include any vesselssuitable for two-phase (gas-liquid) contacting, for example,counter-current flow reactors (e.g., with an upwardly-flowing vaporphase and downwardly-flowing liquid phase) or co-current flow reactors(e.g., with upwardly-flowing gas and liquid phases). In such two-phasecontacting vessels, it is possible for the liquid phase to be thecontinuous phase, as in the case of gas bubbles flowing through a movingcolumn of liquid. Otherwise, it is possible for the vapor phase to bethe continuous phase, as in the case of a dispersed liquid (e.g., in theform of droplets) flowing through a vapor space. As in the case of acirculated loop reactor, different zones of a bioreactor may be used tocontain a continuous liquid phase and a continuous gas phase.

Specific examples of bioreactors include Continuous Stirred TankReactors (CSTRs), Immobilized Cell Reactors (ICRs), Trickle Bed Reactors(TBRs), Moving Bed Biofilm Reactor (MBBRs), Bubble Columns, Gas LiftFermenters, and Membrane Reactors such as Hollow Fiber MembraneBioreactors (HFMBRs). Suitable bioreactors may include static mixers, orother vessels and/or devices (e.g., towers or piping arrangements),configured for contacting the gaseous CO-containing substrate with theliquid bacterial culture medium (e.g., with dissolution and masstransport kinetics favorable for carrying out the biologicalconversion). The phrases “plurality of bioreactors” or bioreactors thatmay be included in a “plurality of bioreactor stages” are meant toinclude bioreactors of more than a single type, although in some casesthe plurality of bioreactors may be of one type (e.g., circulated loopreactors).

Some suitable process streams, operating parameters, and equipment foruse in the biological processes described herein are described in U.S.patent application Publication No. US2011/0212433, which is herebyincorporated by reference in its entirety.

Certain embodiments relate to gas testing units, comprising a firstbioreactor stage for evaluating the performance of a test C1-containingsubstrate and a second bioreactor stage for evaluating the performanceof a reference C1-containing substrate. An analytical section isconfigured for analysis of both gaseous and liquid products of the firstand second bioreactors. The gas testing unit is housed, or at leastcapable of being housed, within a container generally having a volume ofless than about 6 m³ (e.g., from about 0.5 m³ to about 6 m³), typicallyless than about 3 m³ (e.g., from about 1 m³ to about 3 m³), and oftenless than about 2.5 m³ (e.g., from about 1.5 m³ to about 2.5 m³). Inview of such size constraints, the gas testing unit is transportable tomultiple locations, e.g., for evaluating a test (or local) C1-containingsubstrate and optionally other local additives, such as a local watersource. According to further representative embodiments, the gas testingunit is housed, or at least capable of being housed, within a box orother container having length, width, and height dimensions of less thanabout 1.8 meters each (e.g., each of these dimensions being within arange from about 1.0 meters to about 1.8 meters), or less than about 1.6meters each (e.g., each of these dimensions being within a range fromabout 1.0 meters to about 1.6 meters). Such a box or other container mayhave one of its length, width, and height dimensions being less thanabout 1.6 meters (e.g., within a range from about 1.0 meters to about1.6 meters), and the other two of these dimensions being less than about1.3 meters (e.g., within a range from about 0.8 meters to about 1.6meters).

Other embodiments relate to methods for evaluating the suitability of atest C1-containing substrate for use in a bioconversion process. Themethods comprise (a) feeding a reference C1-containing substrate to afirst (reference) bioreactor containing a first culture of a C1-fixingmicroorganism and (b) feeding the test C1-containing substrate to asecond (test) bioreactor containing a second culture of a C1-fixingmicroorganism. The methods further comprise (c) analyzing both gaseousand liquid products of the first and second bioreactors to determine theperformance of the first and second bioreactors. The suitability of thetest C1-containing substrate is established from a comparison of theperformance of the first bioreactor, relative to the performance of thesecond bioreactor. Preferably, at least a portion of the steps (a) and(b) above are carried out simultaneously (i.e., at least a portion ofthese steps overlap in time). Typically, steps (a) and (b) are carriedout simultaneously (or at least substantially simultaneously), for asimultaneous operating period, or test period, of several days (e.g., atleast about 3 days, such as from about 3 to about 21 days; at leastabout 5 days, such as from about 5 days to about 21 days; or at leastabout 7 days, such as from about 7 days to about 14 days), in order toassess the performances of the microorganism cultures in carrying outthe biological conversion process. According to one embodiment, forexample, the entirety of the duration of step (b), in which the testC1-containing substrate is fed to the second bioreactor, may beencompassed by the duration of step (a), in which the referenceC1-containing substrate is fed to the first bioreactor. This will occur,for example, in the case of commencing the operation of both the firstand second bioreactors using the reference C1-containing substrate,followed by changing the feed to the second bioreactor from thereference C1-containing substrate to the test C1-containing substrate.In representative embodiments, therefore, the methods may furthercomprise feeding the reference C1-containing substrate to the secondbioreactor, prior to step (b).

In addition to evaluating test C1-containing substrates, representativemethods may alternatively, or in combination, evaluate local additives,such as a local water source, using the apparatuses and methodsdescribed herein, by determining or assessing comparative performance.In the case of evaluating water quality, for example, different watersources may be used to prepare and/or supplement (e.g., with freshculture medium) the first and second bacterial cultures. According toone embodiment, local conditions may be evaluated by using a local watersource (e.g., local process water or local potable water) to prepare andsupplement (e.g., in the fresh culture medium added) the bacterialculture of the second bioreactor, in combination with feeding the testC1-containing substrate to this culture. In another embodiment, the samelocal water source may be used to prepare and supplement the bacterialcultures of both bioreactors, such that the test C1-containing substrateitself can be evaluated against a baseline using the same water source.In yet other embodiments, the same C1-containing substrate (e.g., eitherthe reference or the test C1-containing substrate) may be fed to bothbioreactors, in order to evaluate the effect of different water sourcesalone (e.g., a local process water source or a local potable watersource, compared to a purified water source such as distilled water).

Yet other embodiments relate to methods for determining whether a testC1-containing substrate supports a biological conversion process.Representative methods comprise (a) maintaining separate, first andsecond cultures of a C1-fixing microorganism, each utilizing a referenceC1-containing substrate as a nutrient for producing ethanol and at leastone further metabolite and (b) changing from the reference C1-containingsubstrate, as the nutrient to the second culture, to a testC1-containing substrate. The methods further comprise (c) assessing theperformance of the first culture, relative to that of the secondculture, under the same target operating conditions (e.g., operating setpoints of automatically and/or manually controlled operating parameters,for example bioreactor pH in some cases), but using the different,reference and test CO-containing substrates. The methods furthercomprise (d) in the event of not obtaining a minimum performance deficit(or offset) of the second culture in step (c), confirming that the testC1-containing substrate supports the biological conversion process. Themethods may additionally comprise (e) in the event of obtaining theminimum performance deficit, or a greater performance deficit, in step(c), pretreating, or enhancing the existing pretreatment, of the testC1-containing substrate to provide a higher quality test C1-containingsubstrate, relative to the test C1-containing substrate used to assessperformance in step (c).

According to alternative embodiments, step (e) in the above methods maycomprise a remedial measure other than improving the quality of the testC1-containing substrate by pretreating or enhancing the existingpretreatment. Such a remedial measure may, for example, includeimproving the quality of a local additive, such as a local water source,or otherwise substituting a higher quality additive, for example, localpotable water for local process water. Other remedial measures mayinclude adjustments of operating conditions, such as bioreactortemperature, pressure, and/or pH. Any type of remedial measure may beaccompanied by re-inoculation of the second bioreactor with a bacterialculture (e.g., a third culture), followed by assessing the performanceof the first culture (or other culture utilizing the referenceC1-containing substrate as a nutrient, or other reference condition)relative to that of the re-inoculated culture for testing the remedialmeasure (e.g., under the same set of target operating conditions, butusing the different, reference and higher quality test C1-containingsubstrates, and/or using the different reference and higher qualityadditive, and/or using an adjusted operating condition). In the event ofnot obtaining the minimum performance deficit of the re-inoculatedculture (e.g., the third culture), then the methods may further compriseconfirming that the remedial measure (e.g., the higher quality testC1-containing substrate, and/or the higher quality additive, and/or theadjusted operating condition) supports the biological conversionprocess. In this manner, a number of remedial measures (e.g.,progressively more highly purified C1-containing substrate) may beassessed, for example in a sequential manner, using the gas testingunits described herein. According to some embodiments, thetesting/evaluation methods may be complete when it isestablished/confirmed that at least one test C1-containing substratequality, additive quality, and/or set of operating conditions supportsthe biological conversion process.

FIG. 1A depicts a side, cut-out view of a representative gas testingunit 1 having both a rear, “wet” or bioreactor stage-containing section200 and a front, “dry” or analytical section 300. Preferably, thesesections 200, 300 are separated by a barrier, such as vertical partition250 that prevents or at least hinders the ambient environmentssurrounding the equipment housed in these sections from intermixing. Arepresentative bioreactor 100 of a bioreactor stage in section 200 has areactor volume generally in the range from about 0.25 to about 5 liters,and often from about 1 to about 3 liters. A typical length of abioreactor, which holds this reactor volume (i.e., which contains thereactor gas and liquid phase contents), is from about 0.5 to about 1.5meters. Normally, bioreactor-containing section 200 will include twoseparate bioreactor stages, as is more apparent from FIG. 1B, for thesimultaneous evaluation of the performance of both reference and testCO-containing substrates.

A bioreactor stage in section 200 may further include an external liquidrecycle loop 25 and an associated external recycle (or recirculation)pump 30 for improving mixing/uniformity within a given bioreactor 100and/or improving the rate of vapor-liquid mass transfer. Using externalliquid recycle loop 25, liquid product, including culture medium and aC1-fixing microorganism, may be withdrawn from a bottom section (i.e.,proximate a bottom end) of bioreactor 100 (e.g., from below a gasdistribution device, such as a sparger and/or from below a liquid inletor a liquid outlet) and recycled externally to a top section (i.e.,proximate an opposite, top end) of the bioreactor 100 (e.g., to above agas/liquid interface that demarcates a boundary between a continuous gasphase zone and a continuous liquid phase zone). As described above,external liquid recycle loop 25 preferably operates without the addedcomplexity required for separation and recycle of the C1-fixingmicroorganism, including membrane filtration systems and associatedcleaning procedures. External liquid recycle pump 30 provides theexternal liquid circulation at a desired rate, for example at an optimumtradeoff between energy usage and mass transfer rate improvement. Othercomponents associated with the mounting and control of bioreactor 100may be included within bioreactor stage-containing section 200, forexample shelving 201 and additional equipment external to bioreactor100, such as that required for reactor temperature control (e.g., heattracing and/or a fan for raising or lowering the temperature ofbioreactor 100, as needed).

Analytical section 300 includes gas chromatography (GC) analyzer 301,including first and second chromatography columns 302 a, 302 b,configured, respectively, for analysis of both the gaseous and liquidproducts obtained from bioreactor stage 10. Such a configuration differsfrom the conventional use of high-pressure liquid chromatography (HPLC)for analysis of metabolite concentrations in liquid products. Althoughembodiments of the invention include the use of HPLC for liquid productanalysis, it has been determined that space is advantageously conservedif the total analytical requirements of the gas testing unit areconsolidated into a single GC analyzer. Generally, the columns used foranalysis of the gaseous and liquid products contain different types of astationary phase (e.g., a resin) for performing the desiredchromatographic separations. Other equipment within analytical section300 may include a high purity air generator (“zero air” generator, notshown) for use as a baseline gas source for the GC analyzer 301,enclosed electrical components 303, and operating software with thenecessary display interface 304 (e.g., a computer), and a utility box305. A satellite communication system 315 may also be included, fortransferring data from the gas testing unit 1, particularly when in useat a prospective installation site with poor or unreliable communicationservice, to a second facility that may be remote from the site (e.g., atleast 100 miles, at least 1,000 miles, or even at least 5,000 miles,away from the site). For example, the second facility may be thedeveloper or licensor of the biological conversion process, having aninterest in the operation of the gas testing unit in real time.Satellite communication system 315 may, therefore, transmit, to thesecond facility, information for use in providing operating instructionspertaining to the gas testing unit 1, such as recommended operatingparameter adjustments or changes in, or the addition of, certainprocessing steps (e.g., gas pretreatment). According to otherembodiments, satellite communication system 315 may allow direct controlof the operation of gas testing unit 1, including the various operatingparameters described herein. Further auxiliary components such as bottomdrawers 306, benches (not shown), and/or a grille fan (not shown), mayalso be included in analytical section 300.

As shown in FIG. 1A, the components of gas testing unit 1 are housedwithin container 500, rendering it easily transportable to a remotelocation for on-site evaluation of a specific C1-containing substrate.This transportability advantageously avoids the potentially misleading(and costly) inaccuracies inherent in attempts to reproduce commercialgas streams, in terms of both composition and fluctuations incomposition, at the site of a fixed laboratory, pilot plant, ordemonstration unit. The depiction of an average size human 600 providesa representation of the typical dimensions of container 500. At the topcovering analytical section 300, container 500 may have a connectionsuch as a hinge connection 550 for opening the container to allow betteraccess to equipment with analytical section 300. It should beappreciated that container 500 need not completely enclose gas testingunit 1, and openings, such as those needed for the operation of exhaustfans, may be provided in container 500. To the extent that container 500includes open or exposed areas (or areas that may be opened), gastesting unit 1 is otherwise at least capable of being housed within acompletely closed container having dimensions as described above. Asshown in FIGS. 1A and 1B, container 500 may be transported on pallet 575and moved relatively short distances via a forklift truck, engaging withforklift receiving openings 590.

The rear cut-out view of FIG. 1B illustrates two bioreactors 100 a, 100b, of respective bioreactor stages 10 a, 10 b, for evaluating theperformance of reference and test C1-containing substrates,respectively. Each of these bioreactors may be equipped with externalliquid recycle loops 25, as described above with respect to FIG. 1A.FIG. 1B, therefore, provides a more complete view of the “wet” orbioreactor stage-containing section 200. In addition to, or alternativeto, the operating control system used for regulating reactor temperature(described above), other operating control systems may be at leastpartly within section 200, although the instrumentation softwareassociated with feedback control loops may preferably be included withinanalytical section 300. Such additional operating control systems may beused for controlling operating parameters such as fresh culture mediumaddition rate, gaseous C1-containing substrate feed rate, and reactorpH. In the case of controlling the surfactant addition rate, variablerate pumps 202 a, 202 b (e.g., syringe pumps) may be used forindependently feeding surfactant to bioreactors 100 a, 100 b. In thecase of controlling the gaseous C1-containing substrate feed rate,appropriate flow control valves may be used, which are sized accordingto the desired gas flow rate and the contemplated pressure upstream ofthe valve (supply pressure) and downstream of the valve (operatingpressure). In the case of controlling reactor pH, the amount of a basicneutralizing agent introduced to a bioreactor stage (e.g., into recycleloop 25, shown in FIG. 1A) may be controlled with variable rate pumps. Arepresentative pH control system is described in greater detail withrespect to FIG. 2.

As shown in FIG. 1B, a total of six pumps are included, with three ofthese pumps 206 a being used to convey basic neutralizing agent andother process liquids (Na₂S, media, etc.) to first bioreactor 100 a, andthe three other pumps 206 b being used to convey such liquids to thesecond bioreactor 100 b. Also housed within bioreactor-containingsection 200 are displays and controllers relating to operatingparameters associated with each bioreactor stage, includingCO-containing substrate flow rate display/controllers 207 a, 207 b,fresh medium flow rate display/controllers 208 a, 208 b, and reactortemperature display/controllers 209 a, 209 b. These displays/controllersmay be included on a fold-down panel (not shown) for ease of operatoraccess/viewing. As described above with respect to the use of asatellite communication system in the analytical section, in analternative embodiment satellite communication system 315 may likewisebe present within bioreactor-containing section 200, with the samefunctionality as described above.

As further illustrated in FIG. 1B, equipment withinbioreactor-containing section 200 includes that associated with directhandling of the feeds that are input to, and the products withdrawnfrom, bioreactors 100 a, 100 b. Examples of such equipment are freshmedia containers 203 a, 203 b and liquid product waste containers 204 a,204 b and their associated connections to bioreactors 100 a, 100 b.Further examples of equipment in this section are bubblers 205 a, 205 bthat may serve various purposes. For example, in a particularembodiment, each bioreactor 100 a, 100 b may have a series of two ormore bubblers in fluid communication with the gaseous products fromthese reactors. It is possible to use one or more empty bubblersdirectly downstream of each bioreactor as a protective measure againstliquid overflow of the bioreactors. One or more fluid-filled bubblersmay be used downstream of the one or more empty bubblers to provide asource of back pressure for diverting gaseous product to the GC when agas sample is to be analyzed. Bioreactor-containing section 200 mayfurther include an exhaust fan and vents (not shown) for allowing thecirculation of fresh air into this section. This can hinder or preventthe accumulation of C1 carbons source (e.g. CO, CO₂, CH₄) in thissection, in the case of leakage, to unsafe levels from the standpoint ofa health risk or an explosion risk. In this regard, a safety controlsystem, comprising a C1 gas detector 210 (e.g. CO detector), may also beincluded in bioreactor-containing section 200. The safety control systemmay be configured to override one or more, for example, all, of theoperating control systems described above (e.g., for controlling thegaseous C1-containing substrate feed rate). For example, the safetycontrol system may suspend the flow of the test C1 containing substrateand/or the reference C1-containing substrate, and preferably both, inresponse to a measurement of an ambient C1 concentration at above athreshold concentration (e.g., an alarm threshold concentration).

FIG. 2 provides further details regarding the operation of bioreactors100 a, 100 b, used for the comparative performance evaluation ofreference and test C1-containing substrates, respectively. According torepresentative processes, these C1-containing substrates are fed to thebioreactor stages through gas inlets 12 a, 12 b positioned proximate thebottom ends of vertically extending bioreactors 100 a, 100 b of eachbioreactor stage. For example, the gas inlets may extend into theirrespective bioreactors within the bottom 25%, and preferably within thebottom 10%, of the length of their respective bioreactors. The gasinlets will normally extend into their respective bioreactors, to gasdistribution devices that may be disposed centrally within thebioreactors at a height corresponding generally to within thesepercentages of reactor length. Particular gas distribution devicesinclude spargers 14 a, 14 b with which the gas inlets may be in fluidcommunication, proximate their respective first ends. Gaseous products,including unconverted C1 carbon source and any gaseous impurities of theC1-containing substrate (e.g., H₂), which are not utilized in thebioconversion reaction, are withdrawn from each bioreactor and exitthrough gas outlets 16 a, 16 b positioned proximate the top ends of thebioreactors, opposite the bottom ends. The gas outlets may extend intotheir respective bioreactors within the top 25%, and preferably withinthe top 10%, of the length of their respective bioreactors, orotherwise, gaseous products may be withdrawn from the tops of theirrespective bioreactors, without the gas outlets extending into theirrespective bioreactors at all.

Liquid product (or “broth”) may be recycled through external liquidrecycle loops 25 a, 25 b for example by pumping, using liquid recyclepumps 30 a, 30 b, from the bottom section of bioreactors 100 a, 100 b,from which liquid product is withdrawn, to the top sections of thebioreactors (e.g., to within the top 10% of the length of bioreactors100 a, 100 b and to above liquid distribution device(s), such as showerheads 110 a, 110 b through which the liquid product is introduced into acontinuous gas phase zone. This liquid then contacts gas that becomesdisengaged at gas/liquid interfaces 22 a, 22 b and continues flowingupwardly (in bulk) through the continuous gas phase zone. In thismanner, bioreactors 100 a, 100 b operate with countercurrent gas andliquid flows (upwardly flowing gas and downwardly flowing liquid) inthis zone, which is disposed above continuous liquid phase zone,operating with internal liquid circulation as described above.

In defining locations of various features with respect to “reactorlength,” this length refers to that of the section containing thereactor contents (an admixture of reactants and reaction products),commonly considered as the “reactor volume,” or “reactor working volume”and this length does not include process lines (e.g., feed inlet linesor product outlet lines) that may extend above or below the reactorvolume, or sections of a column or other vessel that houses a reactorbut does not contain any reactor contents. For example, in the case of acylindrical reactor, the reactor length refers to the length of the axisof the cylinder. The “bottom 10%” of the reactor length refers to arange of heights, starting from the bottom of the reactor and extendingupward for 10% of the reactor length. The “top 10%” of the reactorlength refers to a range of heights, starting from the top of thereactor and extending downward for 10% of the reactor length.

Bioreactors 100 a, 100 b each include liquid inlets 18 a, 18 b, for theintroduction of fresh culture medium and liquid outlets 20 a, 20 b forwithdrawing liquid products of the reactors, which can be sampled todetermine concentrations of ethanol and other metabolites, as well asconcentrations of the C1-fixing microorganism, if desired. The transferof fresh culture medium to, and liquid product (or “broth”) from, eachof the bioreactors 100 a, 100 b, via inlets and outlets 18 a, 18 b, 20a, 20 b, may occur through small bore open pipes (e.g., having innerdiameters from about 1 mm to about 6 mm) in fluid communication withthese inlets and outlets. Liquid products, withdrawn from bioreactors100 a, 100 b, may be passed to, and optionally extend above, height H,corresponding to the working, ungassed liquid level (i.e., a liquidlevel that would exist without gas hold-up). That is, the highestelevation E to which the final stage liquid product extends may be at orabove height H. Height H may be adjustable and may correspondsubstantially to height H of siphon breakers 75 a, 75 b or another typeof liquid take-off point. In the embodiment of FIG. 2, therefore, liquidproduct outlets 20 a, 20 b are in fluid communication with siphonbreakers 75 a, 75 b that are adjustable in height, relative tobioreactors 100 a, 100 b. Elevation E and height H may be regulated togovern the liquid levels or hydraulic heads, i.e., the levels ofgas/liquid interfaces 22 a, 22 b in their respective bioreactors 100 a,100 b, independently.

In the specific embodiment depicted in FIG. 2, liquid inlets 18 a, 18 band liquid outlets 20 a, 20 b are preferably positioned in a quiescentsection below the respective gas inlets 12 a, 12 b and spargers 14 a, 14b, to allow liquid to be fed to, and withdrawn from, this section orreactor location of a given bioreactor stage. It is also possible,however, for inlets and outlets to be positioned elsewhere along thelength of their respective bioreactors, depending on the desiredlocations for the feeding and withdrawal of liquid products. In analternative embodiment, for example, liquid outlets may be positioned ator near the levels of gas/liquid interfaces 22 a, 22 b, for example, toprovide liquid level control based on overflow at the height of liquidwithdrawal.

Conveniently, external liquid recycle loops 25 a, 25 b can providelocations of bioreactor liquid sampling/analysis, and also can beconfigured for bioreactor control. For example, a basic neutralizingagent (e.g., an aqueous base such as an NH₄OH solution or a NaOHsolution) may be added to these recycle loops through basic neutralizingagent inlets 35 a, 35 b as part of an operating control system forcontrolling reactor pH. The operating system can further includeinstrumentation for controlling the flow of the basic neutralizingagent, based on a measured reactor pH, and can more specifically includesuitable feedback control loops associated with each of bioreactors 100a, 100 b. Such control loops comprise, for example, pH analyzers 40 a,40 b that measure (e.g., continuously or intermittently) the pH value ofbioreactor liquid within external liquid recycle loops 25 a, 25 b. Suchcontrol loops also include the requisite hardware (e.g., control valvesor variable rate feed pumps, not shown) and instrumentation software(e.g., computer programs) for comparing the measured pH value to asetpoint value for a given bioreactor, and then controlling the flow ofbasic neutralizing agent to achieve or maintain the set point.

Therefore, external recycle loops of the bioreactors 100 a, 100 b may bein fluid communication with respective basic neutralizing inlets 35 a,35 b and comprise instrumentation for independently controlling pHwithin these bioreactors. External liquid recycle loops 25 a, 25 b mayinclude instrumentation associated with the control of other operatingparameters, such as reactor temperature. For example, temperaturetransmitters that measure (e.g., continuously or intermittently) thetemperature of liquid within the external liquid recycle loops, withsuch temperatures being representative of operating temperatures of thebioreactors, may be used to regulate the operation of heat tracingand/or a fan, described above, for reactor temperature control.Additionally, external liquid recycle loops 25 a, 25 b may includefurther liquid inlets 45 a, 45 b for introducing other liquid diluents,reagents (e.g., surfactants), and/or nutrients, to the bioreactors 100a, 100 b independently at the same or varying rates.

The bioreactor stages 10 a, 10 b may therefore have independentlycontrollable process operating variables, the control of which mayinvolve sampling/analysis of bioreactor liquid product on the externalliquid recycle loops 25 a, 25 b, as described above, and/or theintroduction of fresh culture medium, basic neutralizing agent, and/orother process liquids through any of inlets 18 a, 18 b, 35 a, 35 b, 45a, 45 b. Representative process operating variables include freshculture medium addition rate, gaseous C1-containing substrate feed rate,reactor temperature, reactor pH, and combinations thereof. According tovarious other exemplary control methodologies, (1) the flow of theC1-containing substrate (e.g., the flow of reference C1-containingsubstrate to bioreactor stage 10 a and/or the flow of test C1-containingsubstrate to bioreactor stage 10 b) may be controlled based on themeasured reactor pH, (2) the flow of basic neutralizing agent to eitheror both of bioreactor stages 10 a, 10 b may be controlled based on ameasured acidic metabolite concentration (e.g., acetate concentration)in the corresponding bioreactor liquid product, and/or (3) the flow offresh culture medium to either or both of bioreactor stages 10 a, 10 bmay be controlled based on a measured concentration of the C1-fixingmicroorganism in the corresponding bioreactor liquid product.

The gas testing units described above may be used in methods forevaluating a test C1-containing substrate, for example, available at aprospective installation site for a commercial scale biologicalconversion process. First and second bioreactors may be used forprocessing, respectively, a reference C1-containing substrate (e.g., aC1 carbon source-containing gas of a known composition that may be fixedthroughout the duration of the evaluation method) and the testC1-containing substrate, which may be the available C1-containing wastegas from an industrial facility, such as a steel manufacturing facility,optionally pretreated to remove one or more contaminants. In general,pretreating is performed to remove one or more contaminants of the testC1-containing substrate, or at least a portion of the one or morecontaminants (e.g., at least 75%, at least 90%, or at least 99%, of theone or more contaminants) that are detrimental to the biologicalconversion process (e.g., are harmful to the growth of the C1-fixingmicroorganism). Typically, contaminant(s) in the test C1-containingsubstrate, which are removed by pretreating, are those that, in theabsence of the pretreating, would contribute to an observed performancedeficit in the biological conversion process, when compared to the sameprocess being performed, under the same conditions. Contaminants includehydrocarbons (e.g., benzene) and heteroatom-containing hydrocarbons(e.g., halogenated hydrocarbons or hydrocarbons containing at least oneof Cl, O, N, and/or S, such as dichloropropane, epichlorohydrin, anddioxins). Any of such contaminants are generally present in minoramounts (e.g., in an amount of less than 1%, less than 1000 ppm, lessthan 100 ppm, or even less than 10 ppm, by volume) in the untreated,test C1-containing substrate. Exemplary pretreating includes contactingthe test C1-containing substrate with a solid material or liquidscrubbing medium that selectively removes one or more contaminants, forexample by adsorption or dissolution. Representative solid materialsinclude carbon (e.g., activated charcoal), resins, and zeolites. Othercontaminants include dust particles and other solids (e.g., catalystfines) that may be removed by filtration and/or a liquid scrubbingmedium.

A representative reference C1-containing substrate may be pure CO, or asynthetic blend of CO and one or more other gases (e.g., a CO/H₂ blend,or a CO, CO₂, and H₂ blend). The one or more other gases may be gasesknown to be present in the test C1-containing substrate at approximatelythe same concentrations. A synthetic blend may be representative of acomposition for which performance data has previously been obtained, andoptionally correlated with the performance of a larger-scale operation.In this manner, the comparative performance of the referenceC1-containing substrate with the test C1-containing substrate may beused to calculate a predicted performance of the latter, at thelarger-scale operation, for example, a pilot plant scale, demonstrationscale, or commercial-scale operation. In many cases, a referenceC1-containing substrate, including pure CO or a synthetic blend of CO,may be supplied and fed to one or both of the bioreactors from apressurized cylinder. Using a suitable pressure regulating valve (orseries of valves), the pressure downstream of the cylinder may bereduced to the operating pressure of the bioreactors (e.g., from 0 to 5bar absolute pressure).

The performance of the first and second bioreactors, processing thereference and test C1-containing substrates, respectively, may bedetermined and compared, as a basis for establishing the suitability ofthe test C1-containing substrate. For this purpose, a gas testing unitas described herein may be configured for analyzing both gaseous andliquid products of the first and second bioreactors. For example, thegaseous products may be analyzed to determine the amount of remaining C1gas, following consumption by the bacterial culture, of C1 gas in thereference and test C1-containing substrates. The overall substrateutilization of a bioreactor refers to the percentage of the substratethat is input to that bioreactor and utilized in the conversion todesired product(s) (e.g., ethanol) and other metabolites of themicroorganism. Using a CO-containing gas as an example, if thecomposition of the gaseous product exiting the bioreactor is determined,then the overall CO utilization (expressed as a fraction) may becalculated as:1−(rate of CO exiting the bioreactor)/(rate of CO input to thebioreactor).

The gas testing unit can provide, or can at least provide sufficientinformation (e.g., feed and product gas flow rates and compositions)for, a determination of C1 carbon utilization in each of thebioreactors, as one performance parameter for comparison between thesebioreactors. This C1 carbon source utilization is determined on a “perpass” or “once-through” basis, without accounting for the use of gaseousproduct recycle (and added expense) that can provide higher totalutilization values. However, the per pass C1 carbon utilization can beused in modeling to predict total C1 carbon utilization of a processutilizing such recycle.

Other analytical results from the gas testing unit can be used in thecomparison of performance between bioreactors operating with thereference and test C1-containing substrates. For example, liquidproducts obtained from these bioreactors can be analyzed, typicallyafter separation of the C1-fixing microorganism (e.g., by filtration) todetermine the concentrations (titers) of ethanol and other metabolites,including acetate and 2,3-butanediol. Using the GC analyzer, forexample, all of these concentrations may be obtained in grams per liter,g/l. In some cases, a suitable analytical device may be included withthe gas testing unit, or otherwise used separately for the measurementof C1-fixing microorganism concentration in the liquid product.Representative devices include those measuring the absorbance ortransmission of electromagnetic energy through the sample (e.g., aspectrophotometer), a certain biological activity of the sample (e.g., aplate reader), or another property of the sample (e.g.,impedance/capacitance) in a disposable or reusable probe (e.g., anon-line biomass probe). Analysis of the gaseous and liquid products maybe performed continuously (e.g., using an online analyzer) orintermittently. Analysis may also be conducted automatically ormanually, with manual injection into an analyzer, such as a GC, oftenbeing preferred due to flexibility in sample preparation and a reductionin equipment requirements. For example, sampling systems for theautomated analysis of liquid products of the bioreactors can includesuitable conduits (e.g., tubing or piping), valves, pumps, and actuatorsto allow sampling of the desired bioreactor at the desired time, andsuitable devices for flushing (purging) sample lines to obtain accurateresults. In view of these considerations, and according to particularembodiments, analysis of gaseous products may be performedautomatically, and analysis of the liquid product may be performedmanually.

The analysis of the gaseous and liquid products of the bioreactors overtime allows for the monitoring of one or more performance parameters,used as a basis for establishing the suitability of a given testC1-containing substrate, optionally having been subjected to pretreatingas described above. The comparison of the performance of the firstbioreactor (processing reference C1-containing substrate), relative tothe performance of the second bioreactor (processing test C1-containingsubstrate) may generally involve assessing whether one or more measuredperformance parameters deviates substantially (i.e., exhibits aperformance deficit or offset) with respect to the second bioreactor (orbioreactor culture), relative to the first bioreactor (or bioreactorculture). The performance of the bioreactors may be compared, forexample, over a simultaneous period of operation, or test period, asdescribed herein. To obtain sufficient data regarding the performanceover the operating periods of the first and second bioreactors (i.e.,the time periods over which these bioreactors are fed the reference andtest C1-containing substrates, respectively), gaseous and liquidproducts of the bioreactors may be analyzed, if not continuously, thenintermittently over the respective bioreactor operating periods atsufficient sampling intervals. Representative sampling intervals rangefrom about 15 minutes to about 10 hours and are normally from about 30minutes to about 8 hours. According to a particular embodiment, gaseousproducts are sampled and analyzed at intervals ranging from about 30minutes to about 2 hours, and liquid products are sampled and analyzedat intervals ranging from about 4 hours to about 8 hours. Preferably,gaseous and liquid product samples are taken and analyzed atsubstantially constant intervals, during the bioreactor test periods.

As described above, one performance parameter that may be comparedbetween the bioreactors is C1 carbon source utilization. Otherperformance parameters include the ethanol concentration (titer) in theliquid products of the bioreactors and/or the concentrations of one ormore metabolites (e.g., acetate) in these liquid products. A furtherperformance parameter is the ratio of ethanol to a given metabolite(e.g., the ethanol/acetate weight ratio) in the liquid products.Suitability of a given test C1-containing substrate may be establishedif one or more of these performance parameters are not substantiallydifferent with respect to the second bioreactor (or bioreactor culture),relative to the first bioreactor (or bioreactor culture). The thresholdlevel of difference that may be tolerated, according to someembodiments, can be quantified in terms of a minimum performance deficit(or offset) of the second bioreactor, relative to the first bioreactor.

For example, in the case of the performance parameters described above,a performance deficit may be based on the average value of theperformance parameter over the test period (e.g., the average value ofthe C1 carbon source utilization measured in the first bioreactor,compared to that of the second bioreactor). A minimum performancedeficit can be, for example, at least a 5% deficit, at least a 10%deficit, at least a 15% deficit, or at least a 20% deficit in theaverage value of the measured performance parameter. As would beapparent to those skilled in the art, having regard for the presentspecification, other specific minimum performance deficits (e.g., anyvalue in the range from at least a 1% deficit to at least a 75% deficit)can be used to quantify the threshold difference that may be toleratedto establish suitability, depending on the particular performanceparameter and other factors. As would also be apparent to those skilledin the art, having regard for the present specification, a “deficit”refers to a decrease in the performance of the second bioreactor,relative to the first bioreactor, for example (1) a percentage reductionin average C1 carbon source utilization of the second bioreactor,relative to the first bioreactor, (2) a percentage reduction in averageethanol concentration in liquid products of the second bioreactor,relative to those of the first bioreactor, (3) a percentage increase inaverage concentration of acetate or other metabolite in liquid productsof the second bioreactor, relative to those of the first bioreactor, or(4) a percentage reduction in the average ratio of ethanol to a givenmetabolite (e.g., acetate) in liquid products of the second bioreactor,relative to those of the first bioreactor.

According to other embodiments, a performance deficit may be based onthe rate of change of the performance parameter over the test period(e.g., the rate of change of the C1 carbon source utilization measuredin the first bioreactor, compared to that of the second bioreactor), andtherefore the minimum performance deficit can reflect a desired degreeof stability to be achieved using the test C1-containing substrate. Arate of change can be expressed as an average difference in the measuredperformance parameter per unit time (e.g., average % C1 carbon sourceutilization loss/day), or otherwise expressed in terms of a rateconstant obtained from fitting the measured values of the performanceparameter to a model equation, such as an exponential rate equation(e.g., an exponential decay equation, or first order or higher orderreaction rate equation, or kinetic expression). In such cases in which arate of change is used as the basis for a given performance deficit, therepresentative, minimum performance deficits as described above (interms of percentages) are still applicable. Also, in such cases, a“deficit” again refers to a decrease in the performance of the secondbioreactor, relative to the first bioreactor, for example (1) apercentage increase in the average rate of C1 utilization loss, orassociated decay rate constant, of the second bioreactor, relative tothe first bioreactor, (2) a percentage increase in average rate ofethanol concentration loss, or associated decay rate constant, in liquidproducts of the second bioreactor, relative to those of the firstbioreactor, (3) a percentage increase in the average rate of increase inthe concentration of acetate or other metabolite, or associated rateconstant, in liquid products of the second bioreactor, relative to thoseof the first bioreactor, or (4) a percentage increase in the averagerate of decrease in the ratio of ethanol to a given metabolite (e.g.,acetate), or associated rate constant, in liquid products of the secondbioreactor, relative to those of the first bioreactor.

Other performance parameters, or other changes in performanceparameters, may be used as a basis for establishing the suitability of agiven test C1-containing substrate, as would be appreciated by thoseskilled in the art, having regard for the present specification.Normally, the comparison between the performances of the bioreactors,used as a basis for establishing suitability of a test C1-containingsubstrate, comprises measuring at least the concentrations of at leastone C1 carbon source in the gaseous products of the first and secondbioreactors and measuring concentrations of ethanol and at least onefurther metabolite (e.g., acetate) in the liquid products of thesereactors. In many cases, the compositions of the reference C1-containingsubstrate, used for feeding the first bioreactor, is known and thereforenot analyzed on a continuous or even a periodic basis during the testperiod. This may also be true in the case of the test C1-containingsubstrate, at least over some limited duration of operation (e.g.,ranging from about 1 to about 5 days), which may correspond to a testperiod. According to other embodiments, the composition of the testC1-containing substrate may fluctuate significantly during the testperiod, and indeed such fluctuations may be valuable for assessingperformance under a realistic composition range that would beencountered in commercial practice. In particular embodiments, theconcentration of the C1 carbon source or other gases in the testC1-containing substrate may fluctuate by at least about 20% (e.g., fromabout 20% to about 500%), based on 100%×(the highest concentration/thelowest concentration-1), in which the highest and lowest concentrationsare those measured during the test period. In other embodiments, thisdeviation may be at least about 40% (e.g., from about 40% to about250%), of at least about 50% (e.g., from about 50% to about 100%).

According to the specific method depicted in FIG. 3, the biologicalconversion process, for example microbial fermentation for theproduction of ethanol from C1-carbon source using a C1-fixingmicroorganism, is established in both the first and second bioreactorsusing the reference C1-containing substrate (e.g., a synthetic gas,which may be a blend of gases, as described above). In this step, theseparate, first and second cultures of the C1-fixing microorganism aremaintained, utilizing the reference C1-containing substrate as anutrient for both cultures. According to one possible procedure forinitiating the process, the first and second bioreactors may beinoculated or charged with C1-fixing microorganism initially (e.g., infreeze-dried form), and, after a period of batch growth in culture, themicroorganism may achieve a sufficiently high concentration, such thatcontinuous addition of fresh culture medium can be initiated.

In representative embodiments, the culture is established, for exampleby batch and then continuous operation, for a total period of 4 days, ormore generally from about 1 day to about 10 days, for example from about2 days to about 7 days. After this period, the reference C1-containingsubstrate, having been fed to the second bioreactor, is changed to thetest C1-containing substrate (process gas), whereas the referenceC1-containing substrate is continually fed to the first bioreactor.Operation of the first bioreactor is maintained with a stable bacterialculture (“control/seed culture”) that may be used, if necessary, to seedor re-inoculate the second bioreactor, in the event of poor/unstableoperation of this bioreactor. The first bioreactor, having the“control/seed culture,” is operated during the period of assessing theperformance of the second culture, relative to that of the first, e.g.,by comparing one or more of the performance parameters described above,based on analytical results obtained from the gaseous and liquidproducts of the bioreactors.

As shown in FIG. 3, steady-state operation in the second bioreactor,following the switch from reference to test C1-containing substrate, canrequire 3 days, or, more generally, from about 1 to about 7 days. Thetest C1-containing substrate can be fed to this “experimental culture,”under steady state testing (evaluation) conditions, for an additional 3days, or, more generally, for an additional 1 to 7 days of arepresentative testing period. To confirm the suitability of the testC1-containing substrate, an additional period of “stability testing” maybe performed, with the frequency of analysis of the gaseous and liquidproducts being the same as in, or perhaps decreased relative to, that ofthe preceding performance assessment of the “experimental culture.” Theconfirmatory stability testing period may last, in representativeembodiments, from about 3 days to about 28 days, and typically fromabout 7 days to about 21 days. Therefore, the second bioreactor may bemonitored for performance with the experimental culture, during the timethat steady-state conditions are established for this culture, and/orduring the subsequent period of stability testing. In the event thatinstability is encountered during either or both of these periods, or inthe event that a minimum performance deficit, as described above, isobtained (i.e., a minimum tolerable level of deficit of a performanceparameter, as described above, is exceeded), the second bioreactor maybe seeded or re-inoculated. In the event that no instability (or aminimal or tolerable level of instability) is encountered, or in theevent that a minimum performance deficit, as described above, is notobtained (i.e., a minimum tolerable level of deficit of a performanceparameter, as described above, is not exceeded), then it can beestablished or confirmed that the test C1-containing substrate issuitable for the biological conversion process.

If it becomes necessary for the second bioreactor to be seeded orre-inoculated (e.g., with a third bacterial culture) a remedial measure,as described above, may be tested, followed by repeating the steps, withrespect to the second bioreactor, of achieving steady-state operation,conducting performance assessment relative to the first bioreactor,and/or performing stability testing. A representative remedial measureis a higher quality (e.g., purer) test C1-containing substrate, obtainedfollowing enhanced gas treatment (purification), prior to introductionto the second bioreactor. Testing of the remedial measure may involve aperformance assessment based on obtaining the same or a differentminimum performance deficit, as in the original testing. As illustratedin FIG. 3, a period of “further stability testing” may be performed toestablish or confirm that the remedial measure is suitable for thebiological conversion process. Conditions and duration of the furtherstability testing may be the same or different, relative to those of theoriginal stability testing. As will be appreciated by those skilled inthe art, having regard for the present specification, testing offurther, successive remedial measures (e.g., using fourth, fifth, sixth,etc., bacterial cultures) may be performed, particularly in the eventthat prior remedial measures are not successful.

The following examples are set forth as representative of the presentinvention. These examples are not to be construed as limiting the scopeof the invention, as these and other equivalent embodiments will beapparent in view of the present disclosure and appended claims.

Example 1

Unit Configuration

A gas testing unit was constructed, having a bioreactor section,including two circulated loop reactors (2 liters in reactor volumeeach). The control of C1-containing substrates (reference and test) wasbased on gas flow meter/controller settings, and an automatic pHcompensation (control) system was included for each reactor, based onthe adjustment of NH₄OH or other basic neutralizing agent flow. Thereactor stages did not include membrane separation systems for theseparation and recycling of the bacterial culture. Heat tracing and afan were installed in the bioreactor section for reactor temperaturecontrol. Equipment in the analytical section, maintained apart from thebioreactor section using a vertical partition, included a dual columngas chromatograph (separate GC columns for gas and liquid samples) andvalves/actuators to allow for automated sampling for gas analysis. Theanalytical section was configured for manual injection into the GC ofliquid products from the reactors, for determination of theconcentrations of ethanol, acetic acid (acetate), and 2,3-butanediol. Alaptop computer was included in this section for control of theanalytics and process operating parameters.

More specifically, the gas chromatograph was customized with an externaloven, valves, actuators, and a 6-port selection valve to allow for theautomated and continuous analysis of gaseous products. This valve wascontrolled by the software that also controlled the GC and that wasexecuted by the laptop computer. Only the valves and sample holding loopwere configured external to the main GC oven; other column componentswere located in the oven. Segregation of the actuators and valves fromthe oven was chosen as a way to prevent thermal expansion andcontraction and thereby prolong the operating lifespan and reducemaintenance. The width of the GC and its supporting components wasapproximately 80 cm. Other equipment for use with the GC included azero-air generator, a thermal conductivity (TCD) detector (to be runwith high purity pure argon), and a flame ionization detector (FID) (tobe run with compressed air and hydrogen).

The bioreactor and analytical sections (including the GC and itssupporting components, with approximately 10 cm around its periphery toallow cooling) were fit into a self-contained box for transport.Pretreatment of the C1-containing substrate, using for example activatedcarbon, was considered an “outside the box” option, depending on thecustomer's gas quality. For additional control/minimization oftemperature fluctuations, the gas testing unit could be housed indoors(e.g., within a temporary building).

Example 2

The Gas Testing unit of Example 1 was sent to a customer site tofacilitate testing of the customer C1-containing substrate (testC1-containing substrate). The C1-containing gas to be tested was asindustrial gas produced as a major by-product of a phosphorus productionprocess. Typically, the C1-containing as was being flared by thecustomer. The gas testing unit was sent to the site to determine whetherthe test C1-containing substrate was suitable for conversion to productsby a biological conversion process.

The composition of the test C1-containing substrate is shown in table 3.

TABLE 3 Bulk Composition Known Contaminants (ppm) CO N₂ CO₂ H₂ PH₃ H₂SP₂O₅ P₄ 72% 20% 1% 6% 1200-1400 0-1000 1000-2000 300-1000

Gas clean-up of the test C1-containing substrate typically involvedpassing the test C1-containing substrate through an electrostaticprecipitator and water scrubber. The test C1-containing substrate wasfurther treated to remove known contaminants. The further treatmentincluded the use of two foam scrubbers and an activated carbon bed. Thefirst foam scrubber contained a sodium carbonate solution (5%), thesecond foam scrubber contained a copper sulfate solution, and the carbonbed contained approximately 10 kg “sulfisorb 8 GAC” from Calgon.

A compressed air gas booster was used to increase the pressure of thetreated test C1-containing substrate to provide to a minimum of 2.0 bargat the inlet of the gas testing unit.

Three test runs were performed at the customer facility to assess thesuitability of the test C1-containing substrate. Test runs 1 and 2 wereperformed using a treated test C1-containing substrate, and Test 3 wasperformed using a raw/untreated test C1-containing gas.

Test run 1 was performed using a treated test C1-containing substratehaving the following composition:

CO PH₃ (ppm) H₂S (ppm) P₂O₅ (ppm) P₄ (ppm) 60% 1.0-1.8 53 15-60 20

Liquid nutrient media was added to the GTS reactor vessel. The liquidnutrient media contained, per liter, MgCl, CaCl₂) (0.5 mM), KCl (2 mM),H₃PO₄ (5 mM), Fe (100 μM), Ni, Zn (5 μM), Mn, B, W, Mo, and Se (2 μM).The media was autoclaved, and after autoclaving, the media wassupplemented with thiamine, pantothenate (0.05 mg) and biotin (0.02 mg)and reduced with 3 mM cysteine-HCl.

Nitrogen gas is sparged into the reactor vessel, and the pH and ORP areadjusted. The GTS reactor vessel is then inoculated with freeze-driedcells through a syringe. The freeze-dried cells were Clostridium.autoethanogenum strain DSM23693 deposited at DSMZ (The German Collectionof Microorganisms and Cell Cultures, Inhoffenstraße 7 B, 38124Braunschweig, Germany). The input gas is then switched from nitrogen gasto the treated C1-containing gas.

The test run was performed over a period of 5 days. At day 4.2 growth ofthe Clostridium autoethanogenum culture was confirmed visually. At day5, GC analysis of the fermentation broth confirmed an ethanolconcentration of 1.6 g/L and an acetate concentration of 5.4 g/L.

Test run 1 confirmed the successful revival of the freeze-driedinoculum, demonstrated continuous growth of the culture and demonstratedethanol production by the culture. Test run 1 confirmed that the treatedtest C1-containing substrate was suitable for the biological process,and demonstrated that no unknown contaminants, which have a negativeimpact on growth, were present in the test C1-containing substrate.

Test run 2 was performed using a treated C1-containing substrate havinga 13% CO composition. Visual confirmation of growth was confirmed on day3.75. At day 4.75, a relatively stable acetic acid concentration of 5-6g/L was shown, with no concurrent ethanol production. This result isconsistent with undersupplied culture conditions. The CO composition ofthe incoming test C1-containing was increased to a concentration of 72%on day 9.73. Over the next 3 days, ethanol production was observed, withmeasurements of greater than 8 g/L of ethanol observed.

Test run 2 confirmed the findings of Test run 1.

Test run 3 was started using treated test C1-containing gas having a COcomposition of 72%. Once growth of the culture had been determined, thegas was switched to an untreated test C1-containing substrate. Theculture collapsed within a day of the untreated test C1-containing beingsupplied to the gas testing unit. Test run 3 confirmed that theraw/untreated test C1-containing gas is not suitable for the biologicalprocess.

Unit Operation/Auxiliary Equipment

Both bioreactors would be charged (inoculated) with freeze-driedorganisms, and the cultures established using synthetic gas, such ascylinder gas from a local supplier. Following the start-up withsynthetic gas, one reactor would be switched to site gas to validateperformance on stream, for a testing period of several days to severalweeks. If the site gas is not available at sufficient pressure, e.g.,nominally at least about 2 bar absolute pressure, for example in a rangefrom about 3 to about 10 bar absolute pressure, a booster compressor maybe used as needed to increase the available pressure of the site gas tosuch pressures, thereby ensuring a stable input to the bioreactors. Avalve used for switching the source of gas to a bioreactor, fromsynthetic gas (e.g., bottled or cylinder gas) to site gas, may in somecases have additional ports for allowing gas flow from alternativesources. For example, a 3-way valve may allow an operator to alter thesource from among a synthetic gas, site gas, and a purge gas, which maybe an inert gas such as nitrogen. Optional batch runs could be performedto investigate increasing the ethanol titer of the liquid products. Dueto the low projected gas flow rates (on the order of 2 liters/minute)needed for the testing, the gaseous products exiting the gas testingunit could be either returned to their source (e.g., the customer'swaste gas stream) or otherwise vented to the atmosphere.

An exemplary listing of equipment, auxiliary materials, and support tobe included with the gas testing unit, as well as equipment/requirementsof the prospective facility (customer) and further requirements fromlocal vendors, as needed for implementation/operation of the gas testingunit, is as follows:

Included with Gas Testing Unit Prospective Facility Local VendorsCompressor (if required) Site gas at nominal 5 Bar pressure Syntheticgases Microbes Vent (or return to the origin) (N₂, CO, Argon, 1x staffsupport Housing (air-conditioned) Hydrogen) Media (in powder form) WaterAnalytics (GC-MS) Glassware Waste disposal laboratory Chemicals(supplied in Gas treatment (optional) pre-packs) Information regardingsite gas: Gas treatment (optional) composition fluctuations, Operatingcontaminant identity, and Instructions/Manuals concentrations. Biomassprobes CO₂ fire extinguisher (optional) Table General laboratoryconsumables (syringes, tubes, needles, filters, etc.)Capabilities/Objectives/Deliverables

Some key capabilities associated with the operation of the gas testingunit include the verification of (1) stable and otherwise acceptableoperation throughout a range of changing gas compositions supplied bythe facility, (2) positive microorganism growth on untreated gas, (3)the contaminant profile of gas and liquid samples, (4) performancetargets obtained elsewhere (in off-site testing) using a syntheticblend, (5) any operating discrepancies caused by the use of site gasversus synthetic gas and/or the use of process (local, on-site) waterversus tap (local, potable) water and also versus purchased, distilledwater.

Further objectives of the on-site testing of gas from a prospectivefacility are (1) to obtain a comparison of bioreactor performance withsite gas, either with or without pretreating, and process water, versussynthetic gas, (2) to assess the impact of gas contaminants includingtrace compounds, in aggregate, on gas uptake, microorganism growth, andmetabolite selectivity, relative to synthetic gas without contaminants,(3) to assess the impact of site process water, similarly, (4) to assesswhether further gas cleanup/pretreatment is required, (5) to assesswhether local process water will support bacterial growth at variousrates of diluent (fresh medium) introduction, (6) to verify or updatereactor models with the data obtained and thereby improve performanceestimates as a basis for providing guarantees, (7) to obtain a“post-mortem” analysis of gas contaminants, desorbed from gaspretreatment beds (adsorbent beds), for example by comparison to knowncontaminants.

Key deliverables to be provided, as a result of information gained fromthe gas testing unit, were expected to vary and depend on the needs ofthe project and prospective facility. Some representative examples ofdeliverables are verification that (1) the facility provides a gasstream that supports the biological conversion process, (2) product(ethanol) and metabolite selectivity's are acceptable from an economicstandpoint, (3) proposed gas purification strategies (if used) areeffective, (4) process water or another local water source isacceptable, (5) the range of gas composition fluctuations can betolerated and their influence predicted, (6) the GC analyses and otherinformation of the gas testing unit is accurate, (7) gas contaminantlevels (if detected) can be tolerated by the microbial culture.

Overall, aspects of the invention are directed to transportable unitsfor the on-site testing of the actual gas, generated at a prospectivefacility, for use in biological conversion processes, and particularlyfor the microbial fermentation of CO-containing substrates for ethanolproduction. The gas testing units and associated methods andmethodologies for establishing the suitability of a test CO-containingsubstrate provide a number of advantages as described herein,particularly with respect to obtaining realistic and accurateperformance expectations and objectives, which could not otherwise beobtained from attempts to simulate a commercial gas compositionoff-site. Those having skill in the art, with the knowledge gained fromthe present disclosure, will recognize that various changes can be madein the apparatuses and methods described herein, without departing fromthe scope of the present invention.

The invention claimed is:
 1. A gas testing unit, comprising: (i) avertically extending first bioreactor vessel comprising a first gasinlet port and a first gas outlet port, the first gas inlet port locatedproximate to the bottom end of the vertically extending firstbioreactor, the first inlet gas port in fluid communication with acylinder filled with a reference gaseous C1-containing substrate, thefirst inlet gas port extending into the interior of the bioreactorvessel and in fluid communication with a first gas distribution devicelocated within the bioreactor, the first gas outlet port locatedproximate to the top end of the vertically extending first bioreactorvessel in fluid communication with the first bioreactor and configuredto withdraw gaseous products, including unreacted C1-containingsubstrate, from the first bioreactor vessel, the first gas outlet portin fluid communication with piping means, said piping means in fluidcommunication with a first column of a gas chromatograph configured toanalyze the gaseous products; the first bioreactor vessel furthercomprising a first liquid inlet port and a first liquid outlet port, thefirst liquid inlet port positioned below the first gas inlet port andconfigured to introduce a culture medium comprising at least oneC1-fixing microorganism into the first bioreactor vessel, the firstliquid outlet port positioned below the first gas inlet port andconfigured to withdraw liquid products from the first bioreactor vessel;(ii) a vertically extending second bioreactor vessel comprising a secondgas inlet port and a second gas outlet port, the second gas inlet portlocated proximate to the bottom end of the second vertically extendingsecond bioreactorvessel, the second gas inlet port in fluidcommunication with piping means, said piping means in fluidcommunication with an industrial facility and configured to feed a testgaseous C1-containing substrate from the industrial facility to thesecond bioreactor vessel, the second inlet gas port extending into theinterior of the second bioreactor vessel and in fluid communication witha second gas distribution device located within the bioreactor, thesecond gas outlet port located proximate to the top end of thevertically extending second bioreactor vessel in fluid communicationwith the second bioreactor vessel and configured to withdraw gaseousproducts, including unreacted test C1-containing substrate, from thesecond bioreactor vessel, the second gas outlet port in fluidcommunication with piping means, said piping means in fluidcommunication with the first column of the gas chromatograph configuredto analyze the gaseous products; the second bioreactor vessel furthercomprising a second liquid inlet port and a second liquid outlet port,the second liquid inlet port positioned below the second gas inlet portand configured to introduce a culture medium comprising at least oneC1-fixing microorganism into the second bioreactor, the second liquidoutlet port positioned below the second gas inlet port and configured towithdraw liquid products from the second bioreactor vessel; and (iii)the gas testing unit comprising the first bioreactor vessel, the secondbioreactor vessel and the chromatograph are all housed within acontainer having a volume of less than about 6 m³.
 2. The gas testingunit of claim 1, further comprising the first and second liquid outletports in fluid communication with piping means, said piping means influid communication with a second column of the gas chromatograph. 3.The gas testing unit of claim 1, wherein the first and second bioreactorvessels further comprise external recycle loops and recirculation pumps,the recycle loop comprising piping means in fluid communication with thebioreactor vessels through exit recycle ports located proximate to thebottom end of the bioreactor vessels, the piping means being in fluidcommunication with the recirculating pumps configured to pump afermentation broth from the exit recycle ports through second pipingmeans to inlet recycle ports located proximate to the top end of thebioreactor vessels, said inlet recycle ports in fluid communication witha liquid distribution device located inside the top end on thebioreactor vessels.
 4. The gas testing unit of claim 1, wherein thecontainer has length, width, and height dimensions of less than about1.8 meters each.
 5. The gas testing unit of claim 4, wherein thecontainer has length, width, and height dimensions of less than about1.6 meters each.
 6. The gas testing unit of claim 5, wherein thecontainer has one of the length, width, and height dimensions of lessthan about 1.6 meters, and the other two of the length, width, andheight dimensions of less than about 1.3 meters.
 7. The gas testing unitof claim 3, wherein the liquid distribution device is a shower head. 8.The gas testing unit of claim 3, wherein the recycle loops furthercomprise basic neutralizing agent inlet ports in fluid communicationwith a basic neutralizing agent source.
 9. The gas testing unit of claim1, further comprising first and second basic neutralizing inlet ports influid communication with a basic neutralizing agent source, the portsconfigured to feed the basic neutralizing agent to first and secondbioreactor vessels respectively.
 10. The gas testing unit of claim 3,further comprising pH analyzers in fluid communication with the recycleloops and configured to analyze the pH of the fermentation broth. 11.The gas testing unit of claim 1, further comprising a first and secondpH analyzer in fluid communication with the interior of the first andsecond bioreactor vessels respectively.
 12. The gas testing unit ofclaim 1, wherein the first and second gas distribution devices arespargers.
 13. The gas testing unit of claim 1, wherein the first andsecond gas inlets are located within the bottom 25% of the bioreactorvessel.
 14. The gas testing unit of claim 1, wherein the first andsecond gas inlets are located within the bottom 10% of the bioreactorvessel.
 15. The gas testing unit of claim 1, wherein the first andsecond gas outlets are located within the top 25% of the bioreactorvessel.
 16. The gas testing unit of claim 1, wherein the first andsecond gas outlets are located within the top 10% of the bioreactorvessel.
 17. The gas testing unit of claim of claim 3, wherein the firstand second recycle loop inlets are located within the top 10% of thebioreactor vessel.